Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

LOAEL (reproductive toxicity) = 99 mg/kg bw/day - OECD 416 - TNO (2007)


 


Hazards identified by EU Risk Assessment in May 2008:
In a two-generation oral reproductive toxicity study a LOAEL of 99 mg/kg/day is derived for effects on fertility based on a decrease in uterus weight observed in all females in F0 and in high dosed F1 female without histopathological correlate.


However, the registrant in accordance with the study director is of the opinion that the proposed LOAEL for fertility is scientifically not justified for the following reasons: The decrease of the uterus weights in females of the low and mid dose groups of the F0 generation was not accompanied by any microscopic findings in the uterus and no effect on fertility was seen in the low and mid dose. The effect may thus be judged as maternal toxicity but not as adverse with regard to fertility. In the high dose group the decerease in uterus weight was accompanied by a slightly abnormal cycle length, but no treatment related microscopical findings were observed. The authors of the study conclude therefore that ‘based on the effects on uterus weight in the F0-female animals, the low dose is considered to be the minimum effect dose for maternal toxicity. Based on the decreased number of pups delivered and the lower pup weight in the mid-dose group of the F1-generation, the low-dose was considered to be the no adverse effect level (NOAEL) for reproduction and developmental toxicity.’

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed to Guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld
- Age at study initiation: (P) 5-7 wks; (F1) 3 wks
- Weight at study initiation: (P) Males: 125-180 g; Females: 82-108 g; (F1) Males: 44-73 g; Females: 41-70 g
- Fasting period before study: N/A
- Housing:premating 3/4 per sex per cage; mating 1male and 1 female; mated females housed singly.
- Use of restrainers for preventing ingestion (if dermal):NA
- Diet (e.g. ad libitum): Rat and Mouse Breeding DIet, RM3, ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period:5 days post quarantine


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24C
- Humidity (%): 40-70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark


IN-LIFE DATES: From: 3 Jul 2006 To: 12 Apr 2007
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
In each dose group, the concentration of the test substance was adjusted over the course of the study to maintain target dose of 0, 100, 333 and 1000 mg TCPP/kg bw/day The animals were fed diets containing the test substance from the start of the study, during the premating period of at least 10 weeks, throughout gestation and lactation until sacrifice.
Details on mating procedure:
- M/F ratio per cage:1:1
- Length of cohabitation: upto 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged singly
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
stability, homogeneity and content of the test substance in the diet were conducted using gas chromatography with flame ionisation detection.
Duration of treatment / exposure:
The animals were fed diets containing test substance from the start of the study, during the premating period of at least 10 weeks, during gestation and lactation until sacrifice. Dams were allowed to raise one litter. At the end of the lactation period pups were weaned and selected for the next generation or sacrificed. F0-and F1-dams were sacrificed at or shortly after weaning. F0- and F1- males were sacrificed after at least 11 weeks of exposure (for sperm analyses and necropsy).
Details on study schedule:
Vaginal smears were made three weeks prior to mating to evaluate the length and normality of the oestrus cycle and daily during the mating period to determine if sperm was present. Upon evidence of copulation, the females were caged individually for the birth and rearing of pups until PN21 or shortly thereafter when they were sacrificed. Dams were allowed to raise one litter per generation. On PN4, litters of more than 8 pups were adjusted to 4 males and 4 females per litter, where possible. On PN21, the litters were weaned and 28 males and 28 females were selected at random from as many litters as possible in each group to rear the next generation. Animals were observed for clinical signs, and food consumption and body weight gain was recorded. Fertility and reproductive performance were measured. F0 and F1 dams were sacrificed at or shortly after weaning. F0 and F1 males were sacrificed after at least 11 weeks of exposure.
Dose / conc.:
85 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
99 mg/kg bw/day (actual dose received)
Remarks:
females
Dose / conc.:
293 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
330 mg/kg bw/day (actual dose received)
Remarks:
females
Dose / conc.:
925 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
988 mg/kg bw/day (actual dose received)
Remarks:
females
No. of animals per sex per dose:
28 animals /sex/dose
Control animals:
yes, plain diet
Details on study design:
The overall intake of TCPP was 0, 85, 293 and 925 mg TCPP/kg bw/day for males and 0, 99, 330 and 988 mg TCPP/kg bw/day for females, for the control, low, mid and high dose groups, respectively.
Positive control:
none
Parental animals: Observations and examinations:
General clinical observations were performed twice daily.
Body weight were recorded at day 0 and weekly throughout the study. Mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 1, 4, 7, 14 and 21of lactation.
Food consumption of males was measured weekly, the consumption by females was measured weekly during premating and pregnanacy and on day 1, 4, 7, 14 and 21 of lactation.
Oestrous cyclicity (parental animals):
Vaginal smears were made three weeks prior to mating to evaluate the length and normality of the oestrus cycle
Sperm parameters (parental animals):
At scheduled necropsy, epididymal sperm was assessed for motility, count and morphology and a testicular sperm count was also made.
Litter observations:
Dams were allowed to raise one litter per generation. Pup body weights, clinical signs and malformations were recorded on days 1, 4, 7, 14 and 21 of lactation. On PN4, litter sizes were adjusted to 4 males and 4 females per litter, where possible. On PN21, the litters were weaned and 28 males and 28 females were selected at random from as many litters as possible in each group to rear the next generation. After selection of pups for next generation, 1 male and 1 female F1 pup of each litter were subjected to a thorough necropsy. After necropsy, the thoracic part of the skeletons was stained and the ribs and sternum of these pups were examined for skeletal abnormalities. For F2 pups, the anogenital distance was measured in all pups on PN1. 1 male and 1 female F2 pup per litter was selected for assessment of vaginal opening and preputial separation.
Postmortem examinations (parental animals):
F0 and F1 dams were sacrificed at or shortly after weaning. F0 and F1 males were sacrificed after at least 11 weeks of exposure.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
There were no treatment related clinical signs in parental animals in either generation. During premating, an F1 male of the mid dose group was found dead on Day 41 and female of the same group was killed in moribund on Day 50. The cause of death or cause of the moribund condition could not be detected at necropsy. There were no other mortalities. A treatment related decrease in body weights was observed in F0 and F1 males of mid and high dose groups, with a larger decrease observed in the F1 generation. During premating, there was no effect on body weight in females of F0 generation but body weights of females in F1 generations were decreased in the mid and high dose groups. During gestation, the mean body weights were decreased in high dose females in F0 females and in mid and high dose F1 females. Body weights were decreased in mid and high dose F1 females during lactation. Mean food consumption was decreased in F0 and F1 males and females of the high dose group and in F0 males and females and F1 females of the mid dose group.
The mean length of the longest oestrus cycle was statistically significantly increased in all dosed F0 females and in high dose F1 females. The number of cycles per animal was significantly decreased in the high dose groups of both the F0 and F1 generations, and the number of acyclic animals was increased in high dose F0 animals only.
This effect on the oestrus cycle appears only to be toxicologically significant at the highest dose as the effect on cycle length was only consistently seen in both the F0 and F1 generations at the highest dose, and is only outside the historical control range at this top dose and the number of acyclic animals and mean number of cycles was only affected in the high dose group. Table 4.48 below summarises the oestrus cycle data.

Table 4.48 Effect of TCPP on oestrus cyclicity
Dose Group
Effect Generation 0 Low Mid High Historical control range$
No. of acyclic females F0 1 0 0 6** -
F1 1 0 1 3 -
Length of longest oestrus cycle (days):
4 F0 18 11 6 1 -
F1 11 10 11 2 -
5 F0 7 13 16 13 -
F1 12 12 7 10 -
6 F0 2 3 5 3 -
F1 4 5 5 8 -
=7 F0 0 1 1 5 -
F1 0 1 3 5 -
Mean F0 4.4 4.8* 5.1** 5.6*** 4.1 ¿ 5.2 (n=15)
F1 4.7 4.9 5.0 5.8***
Mean no. cycles per animal F0 3.9 3.7 3.6 3.0* -
F1 3.8 3.6 3.7 3.1* -
*/**/*** statistically significantly different to the control group p< 0.05/ 0.01/ 0.001. $ Historical control data taken from one-and two-generation oral reproductive toxicity studies and 90-day studies in Wistar rats conducted at TNO between 2003 and 2007

All females, except one of the high dose group in F0, were found sperm positive. One female in low dose group in F0 and one of high dose group in F1 showed only implantations. In both generations, no treatment related differences were observed in pre-coital time, mating index, female fecundity index, male and female fertility index, duration of gestation and post-implantation loss. All dams survived the delivery and there were no dams with stillborn pups in any of the groups.
No treatment related effect on epididymal sperm motility or sperm count, sperm morphology or mean testicular sperm count was observed in either generation at necropsy. Terminal body weights were decreased in mid and high dose males of the F0 generation, in mid and high dose F1 males and in mid dose and high dose females of the F1 generation.
In males, absolute brain weight was decreased in high dose F0 and mid and high dose F1 animals, and relative brain weight was increased in high dose F0 and F1 animals. Relative adrenal weight was increased in high F1 males. Absolute kidney weights were decreased in high dose F0 males and in all dosed F1 males, with relative weights increased in high dose F1 males. Relative liver weights were increased in all dosed F0 males and mid and high dose F1 males. Absolute spleen weight was decreased in high dose F0 males and mid and high dose F1 males. Relative thyroid weights were increased in high dose F0 & F1 males. Decrease in absolute pituitary weight in high F1 males. There was a decrease in absolute epididymal weight in high dose F0 males and an increased in relative weight in high dose F1 males. Absolute seminal vesicle weights were decreased in mid and high dose F0 and F1 animals. Absolute testes weights were decreased in high dose F0 males. Relative testes weight increased in mid and high dosed F1 males. Decrease in absolute prostate weight in high F1 males.
Overall, with respect to effects on organ weights in males, the effect on the kidney at the highest dose group is considered to be the main effect.
In females, absolute and relative liver weights were increased in high dose F0 females and relative liver weight increased in high F1 females. Absolute and relative pituitary weight was decreased in high dose F0 females, in low and high dose F1 females; absolute weight was decreased in mid dose F1 animals. Absolute ovary weight was decreased in high dose F0 females. Absolute and relative spleen weight was decreased in mid and high dose F0 females and high dose F1 females. Absolute brain weight was decreased and relative brain weight increased in high dose F1 females. Absolute kidney weight was decreased in high F1 females.
Absolute and relative uterus weights were decreased in all dosed F0 females and high dose F1 females.
Overall, as regards effect on organ weights in females, there are clear effects on the spleen and the pituitary at the highest dose. The most significant observed in females was a decrease in uterus weight, which was noted at all dose levels of F0 and in the high dose group of F1: 82%, 68% and 68% of the control values for low, mid and high dose groups of F0 generation and 81%, 80% and 65% of the control for the low, mid and high dose groups of F1 generation, respectively. The decrease at the low and mid doses of F1 did not reach statistical significance. It is noted that a decrease in uterus weight was also observed in all dose groups in the preliminary study.
It is noted that the decrease in uterus weights, while significant was not accompanied by any histopathological changes. The oestrus cycle stage was not recorded at necropsy. It is accepted that uterine weight can fluctuate during the oestrus cycle and therefore, there is a possibility that the effects observed may be due to normal variation in uterus weight in cycling females. However, as a reasonable precautionary approach it cannot be excluded that the effects on uterus weight are treatment related. Tables 4.49 and 4.50 below summarises the significant organ weight effects.
Table 4.49 Mean terminal body weights and significant organ weights for males of F0 and F1 generations
Dose Group
Organ Generation 0 Low Mid High
Mean terminal body weight
F0 416.5 400 394.9* 374.1#
F1 397.8 390.8 367.3** 336.1#
Mean absolute organ weight (g)
Kidney F0 2.406 2.333 2.326 2.252**
F1 2.313 2.200* 2.113# 2.061#
Spleen F0 0.742 0.730 0.703 0.629#
F1 0.751 0.736 0.672# 0.596#
Pituitary F0 0.014 0.014 0.013 0.013
F1 0.015 0.015 0.014 0.013#
Seminal vesicles F0 1.595 1.518 1.419* 1.388*
F1 1.475 1.392 1.211# 1.191#
Mean organ weights relative to terminal body weight (g/kg bw)
Kidney F0 5.788 5.850 5.901 6.026
F1 5.843 5.645 5.761 6.164*
Spleen F0 1.781 1.823 1.782 1.683
F1 1.894 1.886 1.834 1.784
Pituitary F0 0.033 0.035 0.032 0.036
F1 0.039 0.038 0.038 0.038
Seminal vesicles F0 3.841 3.808 3.591 3.723
F1 3.712 3.585 3.310 3.511
*/**/# statistically significantly different to the control group p< 0.05/ 0.01/ 0.001


Table 4.50 Mean terminal body weights and significant organ weights for females of F0 and F1 generations
Dose Group
Organ Generation 0 Low Mid High
Mean terminal body weight
F0 267 268 263 258
F1 264 265 251* 246**
Mean absolute organ weight (g)
Liver F0 13.608 13.580 13.702 14.890**
F1 13.629 13.673 13.389 13.872
Spleen F0 0.508 0.490 0.466** 0.443***
F1 0.507 0.505 0.483 0.438***
Pituitary F0 0.016 0.016 0.016 0.015***
F1 0.017 0.015** 0.016* 0.014***
Uterus F0 0.46 0.375* 0.313*** 0.311***
F1 0.455 0.369 0.367 0.295***
Ovary F0 0.082 0.081 0.077 0.073**
F1 0.084 0.080 0.083 0.076
Mean organ weights relative to terminal body weight (g/kg bw)
Liver F0 50.918 50.791 52.031 57.611***
F1 51.590 51.601 53.394 56.202**
Spleen F0 1.9 1.833 1.770** 1.711***
F1 1.922 1.908 1.928 1.779*
Pituitary F0 0.062 0.060 0.061 0.057*
F1 0.065 0.057** 0.062 0.059*
Uterus F0 1.723 1.408* 1.192*** 1.202***
F1 1.732 1.399 1.465 1.202**
Ovary F0 0.309 0.304 0.293 0.285
F1 0.317 0.302 0.331 0.307
*/**/*** statistically significantly different to the control group p< 0.05/ 0.01/ 0.001

There were no treatment related macro-or microscopical changes were observed in the F0 or F1 parental animals. The incidence of mineralisation in the kidneys of the high dose F1-females was higher than in the controls (5/28 in control versus 11/28 in the high dose group). However, kidney mineralisation is a common finding in female rats and therefore not thought to be treatment related. Only the relative liver weight was increased in low dose males and was not accompanied by any increase in absolute organ weight or clinical chemical effects. Therefore, this can be considered an adaptive effect and therefore not adverse.
Key result
Dose descriptor:
LOAEL
Remarks:
parental toxicity
Effect level:
ca. 99 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other:
Remarks on result:
other: Generation: parental (F0 and F1) (migrated information)
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
ca. 85 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: based on decreased body weights, food consumption and organ weight changes.
Remarks on result:
other: Generation: parental (F0 and F1) (migrated information)
Key result
Dose descriptor:
LOAEL
Remarks:
precautionary for fertility
Effect level:
ca. 99 mg/kg bw/day
Sex:
female
Basis for effect level:
other: As for the systemic toxicity EU RAR took this LOAEL as a resonable precautionary level for risk characterisation based on the effects on uterus weight in the F0 generation.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
99 mg/kg bw/day (actual dose received)
System:
other: In the EU Risk Assessment Report the dose of 99 mg/kg bw/day is taken as a resonable precautionary LOAEL for risk characterisation based on the effects on uterus weight in the F0 generation.
Organ:
uterus
Treatment related:
yes
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
The mean length of the longest oestrus cycle was statistically significantly increased in all dosed F0 females and in high dose F1 females. The number of cycles per animal was significantly decreased in the high dose groups of both the F0 and F1 generations, and the number of acyclic animals was increased in high dose F0 animals only. For further information see description of results for P0.
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
The mean number of pups delivered was decreased in the mid-dose group of the F1-generation and in the high dose groups in both generations. This resulted for both high dose groups, in a lower mean number of live pups on PN1 and 4. The effect seen in the high dose group of the F1 generation was mainly due to one litter (10 pups) of dam D597 which was lost entirely on PN4. The study report states that due to a deviation from the study plan, the corpora lutea were not counted at scheduled sacrifice. It is not clear whether the effect on the number of pups per litter on PN1 is due to decreased fertility of the parental animals or a developmental effect on the pups. Additionally, it is noted that the effect on the mean number of pups delivered corrolates with a decrease in maternal body weight observed during the gestation period in these dose groups and therefore may be possibly due to maternal toxicity.
Overall, the effect on the number of pups delivered is observed mainly in the F1 generation, at both the mid and high doses, although the interpretation of the effect at the high dose is hampered by the fact that 10 pups of one single litter died at the high dose. The numbers of pups delivered at the mid and high doses in the F1 generation are outside the historical control ranges. It is noted that there is an increase in post implantation loss in the F1 generation (although this does not reach statistical significance), which could point more towards the observed effect on the number of pups on PN1 being a developmental rather than a fertility effect. Litter data is presented in full in section 4.1.2.9.2
Pup mortality (PN1-4) was statistically significantly increased in the low dose group of F0 and in the high dose groups of F0 and F1. This effect was only observed when the pup was used as the statistical unit. There was no statistically significant difference in the mean number of pups on PN4. Thereafter (up to PN21), all pups of all groups remained alive. Table 4.53 summarises the delivery, pup and litter data.
Table 4.53 Delivery, pup and litter data for F0 and F1 generations
Dose Group
Effect 0 Low Mid High Historical control range$
F0:
Mean no. of pups delivered 10.27 10.67 9.89 9.44* 9.40 ¿ 11.18 (n=19)
Total no. of pups delivered 267 256 277 236
Live birth index (%) 100 100 99 100
No. of pups lost (dying, missing and/ or cannibalized) on:
Days 1-4 3 20*** 10 14**
Days 5-7 0 0 0 0
Days 8-14 0 0 0 0
Days 15-21 0 0 0 0
Mean no. live pups/litter (PN1) 10.27 10.63 9.79 9.44*
Sex ratio on PN1 (M/F) 156/111 129/127 143/134 112*/124
No. pups alive Day 21 198 178 213 190
F1:
Mean no. of pups delivered 10.56 10.00 9.13* 8.68*** 9.40 ¿ 11.18 (n=19)
Total no. of pups delivered 264 240 219 191
Live birth index (%) 100 99 100 100
No. of pups lost (dying, missing and/ or cannibalized) on:
Days 1-4 1 0 2 12***
Days 5-7 0 0 0 0
Days 8-14 0 0 0 0
Days 15-21 0 0 0 0
Mean no. live pups/litter (PN1) 10.52 9.92 9.08** 8.68**
Sex ratio on PN1 (M/F) 140/124 123/117 113106 94/97
No. pups alive Day 21 198 186 181 155
*/**/*** statistically significantly different to the control group p< 0.05/ 0.01/ 0.001
In the F0 generation, the mean number of runts was statistically significantly increased in all dose groups on PN1 and persisted to PN21 in the mid and high dose groups. In F1 generation, the number of runts was increased in the high dose group on PN14 and in all dose groups on PN21. In both generations, the number of runts in the high dose groups increased during the course of the lactation period. Table 4.54 below summarises the number of runts in F0 and F1 generations.

Table 4.54 Clinical observations in pups of F0 and F1 generations on Days 1-21 of lactation
Dose Group 0 Low Mid High
F0
Runts
Day 1 0 14***(7)** 23***(7) ** 11***(3)
Day 4 2(2) 11**(3) 7(5) 6(2)
Day 7 2(2) 13**(3) 20***(7) 21***(6)
Day 14 1 6(2) 15***(7) 26***(9) **
Day 21 1 4(2) 30***(10) ** 97***(19) ***
F1
Runts
Day 1 10(4) 1 17(5) 14(4)
Day 4 4(3) 0 15(3) 16(3)
Day 7 4(3) 2(2) 17(4) 38(8)
Day 14 11(6) 14(3) 19(5) 78***(13)*
Day 21 5(3) 17** (4) 36***(9) 127***(19)***
*/**/*** statistically significantly different to the control group p< 0.05/ 0.01/ 0.001
Figures in brackets represent the number of litters with pups showing the observation

The increased numbers of runts in all dose groups of the F0 generation on PN1 could indicate systemic toxicity to the pups in utero, although it is noted that no similar significant increase in the number of runts was observed in the F1 generation or in the preliminary study at PN1.
One pup of the mid dose group showed a missing eye, which was noticed on PN21.
There was no effect on pup weight at PN1 in either generation. There was no effect on pup weight on PN1 in both generations. Mean pup weights of the high dose group were significantly decreased in F0 generation from PN14 onwards and in the F1 generation from PN 7 onwards. Mean pup weights were decreased in mid dose groups on PN21.
No difference in anogenital distance of the male or female F2 pups was observed between the treated and control animals. Vaginal opening was delayed (not significantly) in the high dose group. Preputial separation was statistically significantly delayed in the high dose group. The mean age of pups reaching these criterion are presented in Table 4.55, below.

Table 4.55 Sexual maturation of F2 pups
Dose Group 0 Low Mid High
Vaginal opening
Pups reaching criteria (%) 92 92 83 80
Day reaching criteria (mean) 39.61 40.77 42.58 46.44
Preputial separation
Pups reaching criteria (%) 96 96 100 100
Day reaching criteria (mean) 43.96 44.13 44.79 47.10#
# Statistically significantly different to the control group p< 0.05/ 0.01/ 0.001

The body weight of the high dose male and females of the F2 generation was significantly decreased from PN28 until PN42 (91% and 89% of control at PN42 for females and males of this group, respectively). The effects observed in this dose group on vaginal opening and preputial separation is most likely secondary to toxicity.
At necropsy of the pups there were no treatment related macroscopic findings. Absolute and relative spleen weights of the F1 and F2 pups of the mid and high dose groups were statistically significantly decreased. No missing 13th rib or cervical ribs were observed in the skeletons of the F1-pups.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
ca. 99 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the decreased number of pups delivered and the lower pup weight in the mid-dose group of the F1-generation
Critical effects observed:
yes
Lowest effective dose / conc.:
330 mg/kg bw/day (actual dose received)
System:
other: decreased number of pups delivered and lower pup weight
Treatment related:
yes
Reproductive effects observed:
not specified

In deriving a N(L)OAEL for effects on fertility, consideration is given to the significant effects observed uterus weight in all dosed females in F0 generation and in high dose animals of F1 generation.

With respect to the decrease in the number of oestrus cycles, this was significant only in the high dose animals and so the effect on the cycle length observed at low and mid doses may be due to normal variation rather than a specific fertility effect.

The study director concluded in the study report that the effects observed on uterus weights in the low and mid dose females of the F0 generation were not adverse since they were not accompanied by any change in the number of oestrus cycles or histopathological findings in the uterus, and that there was no corresponding decrease in uterus weight in the low or mid dose F1 animals.

While the effects on the uterus weight and oestrus cycle may be due to normal variation or weight loss, overall, based on a weight of evidence approach, it cannot be excluded that TCPP has an effect on uterus weight. This effect on the uterus was also observed in all dosed females in the preliminary study. Although the effects on the uterus occurred in the absence of histopathological changes, the magnitude of the decrease in uterus weight in the dosed animals is sufficient to be considered as significant.  In addition, the mean number of cycles per animal are decreased and the length of the longest oestrus cycle are statistically increased in high dose animals of both generations, indicating a possible treatment related effect on the oestrus cycle. Therefore, a LOAEL of 99 mg/kg, based on effects on uterus weight, is derived for effects on fertility.

The low-dose of approximately 99 mg/kg for females is considered to be the LOAEL for parental toxicity. This is based on decreased body weight and food consumption seen in mid and high dose parental animals and the effects on uterus weights seen in all dosed F0 animals. For males, a NOAEL of approximately 85 mg/kg is derived for parental toxicity, based on decreased body weights, food consumption and organ weight changes observed at mid and high dose groups. Based on the decreased number of pups delivered and the lower pup weight in the mid-dose group of the F1 -generation, the low-dose group was considered to be the NOAEL for developmental toxicity.

Executive summary:

In a Two-Generation Reproduction Toxicity Study according OECD Guideline 416, 28 Wistar rats received diets containing 0, low-, mid- and high-dose levels of TCPP ( 84.6 up to 988.2 mg/kg bw) over two successive generations. The animals were fed diets containing test substance from the start of the study, during the premating period of at least 10 weeks, during gestation and lactation until sacrifice. Dams were allowed to raise one litter. At the end of the lactation period pups were weaned and selected for the next generation or sacrified. F0 -and F1 -dam were sacrified at or shortly after weaning. F0 -and F1 -males were sacrified after at least 11 weeks of exposure for sperm analyses and necropsy.

In deriving a N(L)OAEL for effects on fertility, consideration is given to the significant effects observed uterus weight in all dosed females in F0 generation and in high dose animals of F1 generation.

With respect to the decrease in the number of oestrus cycles, this was significant only in the high dose animals and so the effect on the cycle length observed at low and mid doses may be due to normal variation rather than a specific fertility effect.

The study director concluded in the study report that the effects observed on uterus weights in the low and mid dose females of the F0 generation were not adverse since they were not accompanied by any change in the number of oestrus cycles or histopathological findings in the uterus, and that there was no corresponding decrease in uterus weight in the low or mid dose F1 animals.

While the effects on the uterus weight and oestrus cycle may be due to normal variation or weight loss, overall, based on a weight of evidence approach, it cannot be excluded that TCPP has an effect on uterus weight. This effect on the uterus was also observed in all dosed females in the preliminary study. Although the effects on the uterus occurred in the absence of histopathological changes, the magnitude of the decrease in uterus weight in the dosed animals is sufficient to be considered as significant.  In addition, the mean number of cycles per animal are decreased and the length of the longest oestrus cycle are statistically increased in high dose animals of both generations, indicating a possible treatment related effect on the oestrus cycle. Therefore, a LOAEL of 99 mg/kg, based on effects on uterus weight, is derived for effects on fertility.

The low-dose of approximately 99 mg/kg for females is considered to be the LOAEL for parental toxicity. This is based on decreased body weight and food consumption seen in mid and high dose parental animals and the effects on uterus weights seen in all dosed F0 animals. For males, a NOAEL of approximately 85 mg/kg is derived for parental toxicity, based on decreased body weights, food consumption and organ weight changes observed at mid and high dose groups.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
99 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The endpoint is concluded based on a single study assigned a Klimisch rating of 1: reliable without restriction. This has been undertaken according to GLP and an accepted OECD TG for this endpoint (TG416). The database for this endpoint met all relevant data requirements under REACH for the respective tonnage band.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Hazards identified by EU Risk Assessment in May 2008:


"In a two-generation reproductive toxicity study with TCPP, there were no treatment related effects in pre-coital time, mating index, female fecundity index, male and female fertility index, duration of gestation and post-implantation loss. There was no effect on sperm parameters at necropsy. In females, the length of the longest oestrus cycle and the mean number of cycles per animal were statistically significantly increased in high dose animals of both generations. A decrease in uterus weight was observed in all dosed females in F0 and in high dose females in F1. Effects were also noted on pituitary weights, significant in high dose females of both generations. A LOAEL of 99 mg/kg is derived for effects on fertility. This is based on effects on the effect on uterus weight seen in all dosed females in F0 and high dose females in F1."


 


However, the registrant in accordance with the study director is of the opinion that the proposed LOAEL for fertility is scientifically not justified for the following reasons: The decrease of the uterus weights in females of the low and mid dose groups of the F0 generation was not accompanied by any microscopic findings in the uterus and no effect on fertility was seen in the low and mid dose. The effect may thus be judged as maternal toxicity but not as adverse with regard to fertility. In the high dose group the decerease in uterus weight was accompanied by a slightly abnormal cycle length, but no treatment related microscopical findings were observed. The authors of the study conclude that ‘based on the effects on uterus weight in the F0-female animals, the low dose is considered to be the minimum effect dose for maternal toxicity. Based on the decreased number of pups delivered and the lower pup weight in the mid-dose group of the F1-generation, the low-dose was considered to be the no adverse effect level (NOAEL) for reproduction and developmental toxicity.


 


 


Updated relevant information (March 2018):


Tris(chloropropyl)phosphate (M20263) [= TCPP] is on the Testing List of the National Toxicology Program (NTP) with 13-week repeated dose toxicity studies and 2 year carcinogenicitiy studies in rats and mice and a developmental toxicity study in rats. According to NTP the animal experimental phase of these studies is completed. For the carcinogenicity studies histopathology is in progress. Results are not yet available, except for the 13-week study in mice, for which preliminary data in table form (but no organ weights) are shown.


http://ntp.niehs.nih.gov/testing/status/agents/ts-m20263.html


 


As soon as the results of these studies are publically available they will be included in the reproductive toxicity assessment of TCPP. Attention should be focussed iter alia on potential effects on the uterus weight of rats and mice and if the effect seen in the 2-generation reproduction toxicity study will be confirmed or refuted.

Effects on developmental toxicity

Description of key information

Key - NOAEL (developmental toxicity) = at least 500 mg/kg bw/day - OECD 414 - rabbit - van de Ven (2018)


Key - NOAEL (developmental toxicity) = at least 650mg/kg bw/day - OECD 414 - rat - NTP (2020)


Supporting - NOAEL (developmental toxicity) = 99 mg/kg bw/day - OECD 416 - rat - TNO (2007)


Supporting - NOEL (developmental toxicity) = 571 mg/kg bw - prenatal developmental toxicity study - rat - Kawasaki et al (1982)


 


Based on the results of a prenatal developmental toxicity study performed according to OECD 414 on rabbits (2018) following an ECHA Final Decision on a Compliance Check the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the registered substance was established as being at least 500 mg/kg bw/day, the highest dose level tested. A further NTP prenatal developmental toxicity study on rats (2020) established a NOAEL for the registered substance of at least 650 mg/kg bw/day, the highest dose level tested. 

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No OECD Guideline or GLP defined. Study in Janpanese with Tables and Abstact in English
Principles of method if other than guideline:
Fetal toxicities of a flame retardant, tris (chloropropyl) phosphate (TCPP) were studied in Wistar rats.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Route of administration:
oral: feed
Details on exposure:
In the range finding study, groups of 5 female rats were dosed (forcibly by mouth) each day for 7 days with 8, 40, 200 or 1000 mg/kg TCPP suspended in olive oil.
In the main study 11-14 pregnant rats were exposed to a diet containing 1, 0.1 and 0.01% of TCPP from day 0 to day 20 of gesttion.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
no data
Duration of treatment / exposure:
from day 0 to day 20 of gestation
Frequency of treatment:
continuously
Duration of test:
no data
Dose / conc.:
100 ppm
Remarks:
test compound intake: 0.036 g
Dose / conc.:
1 000 ppm
Remarks:
test compound intake: 0.38 g
Dose / conc.:
10 000 ppm
Remarks:
test compound intake: 3.57 g
No. of animals per sex per dose:
100 ppm (13 dams), 1000 ppm (12 dams) or 10000 ppm (14 dams). 11 control dams were used.
Control animals:
yes, plain diet
Details on study design:
Pregnant Wistar rats were administered TCPP in solid food from days 0 to 20 of gestation. Approximately two-thirds of live foetuses were necropsied on day 20 of gestation and examined for skeletal abnormalities, with the remaining third fixed in Bouin's solution and examined for visceral abnormalities. In the post-natal phase, dams were given 0.01 (7 dams), 0.1 (6 dams) and 1% (5 dams) TCPP in the diet up to weaning. 6 control dams were used. Pups were weaned 21 days after birth and monitored until 4 weeks. The litters of dams fed TCPP in the diet throughout pregnancy were adjusted to an average of 8 newborns each within each group and were reared for three weeks with the dams. Table 4.57 below summarises the results obtained following examination of the newborns for abnormalities and growth.
Details on results:
Food consumption and body weight gain among pregnant dams did not differ from controls.
No other effects of TCPP were identified in the dams.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In the rangefinding study, body weight gain was unaffected and no abnormal behaviour or adverse symptoms were recorded. One animal dosed at 1000 mg/kg died on day 2. Kidney weights were significantly increased at 40 mg/kg (10% increase when compared to controls), 200 (increased by 20%) and 1000 mg/kg (increased by 10%). No dose-response effect was observed. Liver weight was also significantly increased at 1000 mg/kg (increased by 10% when compared to control values).
In the main study, food consumption and body weight gain among pregnant dams did not differ from controls. No other effects of TCPP were identified in the dams. Table 4.56 below summarises the effects on the dams and the foetuses on day 20 of gestation.
Dose descriptor:
NOEL
Effect level:
10 000 ppm
Based on:
test mat.
Basis for effect level:
other: no effects were identified in the dams
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no treatment-related effects on foetal mortality, implantation number, resorption or foetal weight. There were no gross abnormalities observed at the birth in any group and there was no difference in the birth rate between the test and control groups. There were no differences between the test and control groups for the weaning rate at three weeks with no abnormalities observed.
Skeletal examination was performed on foetuses from the control and treatment groups. Cervical ribs and missing 13th ribs were encountered in all treatment groups, but not in the control group. 65 control foetuses were examined and none showed cervical ribs. In the 0.01%, 0.1% and 1% treatment groups, 77, 73 and 64 foetuses were examined and 1, 1, and 3 of them showed cervical ribs, respectively. No control foetuses demonstrated missing 13th rib, while 1, 2 and 5 foetuses treated with 0.01%, 0.1% and 1% TCPP showed missing 13th ribs. The incidence of cervical ribs and missing 13th ribs was not reported on a per litter basis and therefore, it is not possible to determine whether the increase in the incidence of these effects was seen only in one litter or spread across a number of litters. Also, due to the relatively low number of foetuses examined, it is difficult to conclude on the dose-dependence and therefore, the significance of the increase in missing 13th rib. Historical control data on the incidence of missing 13th rib was also not available.
Delayed ossification of the sternebrae was seen in 2 foetuses in the control group compared to 3, 7 and 1 foetuses in the 0.01%, 0.1% and 1.0% treatment groups. The authors of the report concluded that these effects were not significant. Following visceral examination of the foetuses only one case of dilatation of the renal pelvis was noted in the 0.1% treatment group. There were no other instances of abnormalities observed in any group following visceral examination. Weaning rate and rearing condition were unaffected by treatment and there was no evidence of any abnormality.

No. of implants and resorptions: 124/12, 135/5, 132/6, 158/8, no effects
No. of dead fetuses: zero thoughout all doses
No. of live fetuses per sex (m/f): 56/56, 67/63, 52/74, 77/73, no effects
Weight of live fetuses: no differences with dose
No. of fetuses with external malformations: zero throughout all doses
No. of fetuses in skeletal examination: 65, 77, 73, 84
cervical rib: 0, 1, 1, 3
short of 13th rib: 0, 1, 2, 5
elayed ossification of sternebra: 2, 3, 7, 1
No. of visceral examinations: 47, 53, 53, 66
orrected dextrocardia: zero throughout all doses
dilatation of renal pelvis: 0, 0, 1, 0
Dose descriptor:
NOEL
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The development of the neonates in all groups was comparable to the control. It is concluded that TCPP possesses no teratogenic effect in rats fed the diet containing ranging from 0.01 to 1% during the whole period of gestation.
Abnormalities:
no effects observed
Developmental effects observed:
no

Calculation of doses (not given in report): about 510, 54, and 5 mg/kg bw/day for the 1, 0.1, and 0.01% groups

Effects of TCPP on foetuses and dams fed from day 0 to day 20 of gestation

Dose (%)

0

0.01

0.1

1.0

No. of animals(dams)

11

13

12

14

No. of implants

124

135

132

158

No. of resorptions

12

5

6

8

No. of dead foetuses

0

0

0

0

Live foetuses:

Male/Female

Male/Female

Male/Female

Male/Female

No.

56/56

63/67

52/74

77/73

Weight (grams)

4.3/4.1

4.4/4.2

4.3/4.1

4.3/4.1

No. of foetuses with ext. malformations

0/0

0/0

0/0

0/0


Table 4.57 Effects of TCPP on neonatal growth

Dose (%)

0

0.01

0.1

1.0

No. of litters

5

6

7

6

At birth:

No. of live neonates

No. of dead neonates

Live birth index (%)

Abnormality of neonates

47

60

74

61

1

3

0

3

89.1

89.4

96

93

0

0

0

0

At weaning:

No. of dead neonates

                       Male:

                       Female:

No. of weanlings

Weanling rate (%)

Abnormality of neonates

1

1

1

0

1

0

0

1

38

47

55

47

95.0

97.9

98.2

97.9

0

0

0

0

Executive summary:

Fetal toxicities of a flame retardant, tris (chloropropyl) phosphate (TCPP) were studied in Wistar rats and the following results were obtained:

1. The oral LD50 was 1.5 g/kg in females.

The relative weight ratio per body weight (100g) of kidney were significantly increased by oral administration of TCPP for 7 days to female rats in both groups of 1000 mg/kg and 200 mg/kg from the control.

2. Teratogenic effects were examined by feeding a diet containing 1, 0.1 and 0.01% of TCPP to 11 -14 pregnant rats from day 0 to day 20 of gestation and at the term, the maternal body weight gain and food intake in all examined groups were almost equal to the control. There were no differences among all the groups in the numbers of implantations, resorptions and fetal body weight. No gross fetal malformations were found in any groups.

3. Skeletal and visceral observations of fetuses indicated that there was no significant difference in frequency of anormalies between the control and the experimental groups.

4. The development of the neonates in all groups (5 -7 litters per group) were equal to the control.

Frome above results, it is concluded that TCPP possesses no teratogenic effect in rats fed the diet containing ranging from 0.01 to 1% during the whole period of gestation.

The NOEL for developmental toxicity was found to be 571 mg/kg bw.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lab specific test material number: 208366/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in refrigerator
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability was analytically confirmed for at least 5 hours at room temperature under normal laboratory light conditions, 13 days in the refrigerator, and 3 weeks in the freezer (≤ -15°C) over the concentration range 1 to 200 mg/mL. Test Facility Study No. 517747 (ABL study no.17117).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Chatillon sur Chalaronne, France
- Untreated females from a non-inbred laboratory colony were mated at the supplier and were at day 0 or 1 post-coitum on arrival at the Test Facility (day 0 post-coitum is the day of successful mating)
- Age at delivery: 18-20 weeks; a health inspection was performed upon receipt of the animals
- Fasting period before study: none
- Housing: individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): relative target humidity of 40 to 70%; the actual daily mean temperature during the study period was 18 to 20°C with an actual daily mean relative humidity of 58 to 90%.
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES Main Study:
From: 29 September 2017 To: 11 October 2017
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous carboxymethyl cellulose with 0.1% Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 5 hours after adding the vehicle to the test item or were prepared weekly by formulating daily portions which were stored in the refrigerator. When prepared weekly, dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water was chosen based on trial formulations performed at Charles River and information provided by the Sponsor.
- Amount of vehicle (if gavage): 5 ml/kg bw
- Preparation of vehicle: The vehicle, 1% Aqueous carboxymethyl cellulose with 0.1% Tween 80, was prepared at least monthly, stored in a refrigerator set to maintain 4°C. The prepared vehicle was removed from the refrigerator and stirred for at least 30 minutes before dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by using a validated analytical procedure (ABL No. 17118).
Concentration analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. The concentrations analysed were in agreement with the target concentrations. No test item was detected in the control group formulation.
Homogeneity analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was below or equal 10%. The formulations of group 2 and 4 were homogeneous.
Stability analysis: Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 517747, ABL No. 17117) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 517747.
Details on mating procedure:
Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).
Duration of treatment / exposure:
Day 6 - 28 post coitum inclusive
Frequency of treatment:
once daily
Duration of test:
All animals surviving to the end of the observation period (Day 29 post-coitum) were euthanised by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol®20%) and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs and the fetuses.
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Each group consisted of 22 mated female rabbits.
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose levels
Dose levels were selected based on the results of a dose range finding study (Test Facility Study No. 517745) and in an attempt to produce graded responses to the test item.
The dose levels for this study were selected based on the results of a tolerability study in non-pregnant rabbits by oral gavage in which no mortality or severe clinical signs became obvious up to and including 1000 mg/kg bw/day (Test Facility Study No. 517744).
In the dose range finding study dose levels of 500, 750 and 1000 mg/kg bw/day were tested in 6 pregnant rabbits per dose and control from gestational day 6 to 28 by oral gavage.
Treatment at 1000 mg/kg bw/day group was poorly tolerated by pregnant females and resulted in early termination of this group. Four out of six females (nos. 19-22) were euthanized in extremis on Days 12 or 13 post-coitum. From treatment onwards (Day 6 postcoitum), these females lost about 280-560 gram body weight (i.e. 9-14%) and had significantly reduced or no food consumption. Moreover, severely reduced feces production piloerection. At necropsy, one female was emaciated and one female had cysts on her ovary and scab formations. Due to animal welfare reasons, the remaining two females (nos. 23-24) of this dose group were sacrificed on Day 13 post-coitum as well.
One female at 750 mg/kg bw/day (no. 16) was euthanized on Day 25 post-coitum as she delivered her offspring early. This female showed a body weight loss of 9% from treatment onwards and had no food consumption over Days 15-24 post-coitum. A lean appearance, reduced feces production, dark urine and alopecia were noted in addition. No abnormalities were observed at necropsy. No toxicologically relevant findings were noted for the remaining animals treated up to 750 mg/kg bw/day.
Based on the results of this dose range finder, in which four animals of the 1000 mg/kg bw/day group and one animal of the 750 mg/kg bw/day group showed severe general toxicity (e.g. severely reduced food consumption, body weight loss) selected dose levels for the main study were 75, 200 and 500 mg/kg bw/day.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 18, 21, 24, 27, and 29 post-coitum

FOOD CONSUMPTION: Yes
- Days 2-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29; females with early delivery (nos. 76 and 83): within 24 hours of early delivery
- Examination at necropsy: All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).
- Organ weight: liver weight
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not for premature decedents)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: pre- and post- implanation loss
Fetal examinations:
Live fetuses were euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube. Fetuses of premature decedents were externally examined in detail and euthanized if necessary by decapitation or by administration of sodium pentobarbital.

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized by administration of approximately 0.3 mL (= 60 mg) of sodium pentobarbital into the oral cavity using a small flexible plastic or metal feeding tube. Recognizable fetuses of females that were killed in extremis were examined externally. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed (if possible). Late resorptions with malformations were fixed in 10% buffered formalin.

Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The sex of all fetuses was determined by internal examination.

The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution. Tissues were then transferred to a 70% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent processing and soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.

All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal:
The eviscerated fetuses from all groups, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. Since no possible treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group.

The specimens of all groups were archived in glycerin with bronopol as preservative.

A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as
indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but
excluded semi-quantitative data, and any group with less than 2 observations.

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
Incidence:
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Maternal variables:
Body Weight Gains: Calculated between at least each scheduled interval.
Corrected Body Weight Gains: Terminal body weight minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Organ Weight Relative to Body Weight: Calculated against the Terminal body weight.

Reproduction and development variables - For each litter the following calculations were performed:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) divided by the number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) divided by the number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%) = number of viable fetuses affected/litter divided by the number of viable fetuses/litter x 100
Historical control data:
historical control data of fetal examination are given for values that are outside the expected range or of statistical significance, as appropriate
Description (incidence and severity):
Clinical signs:
No clinical signs were noted for surviving females that were considered to be toxicologically relevant.
Reduced feces production up to a severe degree was observed for almost all animals in all groups, including controls. This finding is commonly observed for rabbits of this age and strain which are housed and treated under the conditions in this study, and as no dose-related trend was observed, this was not considered to be toxicologically relevant.
A lean appearance, diarrhea, red discoloration of the genital region and red fluid on manure tray were noted for single females across the groups during the treatment period. Restless behavior was noted once for two females treated at 200 mg/kg bw/day, of which one female showed a cramped posture as well, and for one female at 500 mg/kg bw/day. One female at 500 mg/kg bw/day was noted on one single day during the treatment period with rales and a bleeding nose. As these findings occurred incidentally and did not persist over time, they were not considered to be toxicologically relevant.
For clinical signs noted for premature decedents in the low and mid dose group (75 and 200 mg/kg bw/day) see section 'mortality' and Table 1
Description (incidence):
Mortality:
see Tables 1 and 3: Four females of each the low and mid dose group (75 and 200 mg/kg bw/day) died or were euthanized in extremis within a few seconds to minutes after the oral gavage procedure between Day 6 and Day 23 post-coitum.
One female (no. 38) was not dosed on the day of euthanasia as difficulties were noted when inserting the gavage tube. For three females (nos. 38, 53 and 60) blood was noted at the end of the gavage tube. For the other females, no difficulties or abnormalities were noted during the gavage procedure. Clinical signs that were observed directly after the gavage procedure included labored respiration (three females), gasping (two females), clonic spasms (four females), a pale appearance and uncoordinated movements (one single female). In addition, three females were noted with a bleeding nose and/or mouth. These effects were almost directly followed by death or euthanasia. Macroscopic findings were mainly observed in the respiratory tract of these premature decedents. Foamy contents in the trachea were noted for four females. Reddish foci in the lungs (up to many) were noted for seven females, of which two females especially showed foci in the right caudal lung lobe. Three females showed inflation of the lungs. For single females each, a ruptured right caudal lobe, reddish discolored lungs or a perforation of the right caudal lung lobe was noted. Additionally, hemorrhagic and clotted blood in the thoracic cavity or a hardened heart were noted for one female each.
In conclusion, these 8 unscheduled deaths occurred only in the low and mid dose group, and not in the high dose group, and the moribund effects occurred timely very closely related to the gavage procedure, i.e. within a few seconds to minutes. Most of the clinical signs (blood at the end of the gavage tube, gasping, labored respiration) and macroscopic findings in the lungs and trachea can be related to the oral gavage procedure, and for two out of eight females a perforation or ruptured lung lobe were observed at necropsy indicating a misapplication. Therefore, the deaths can be interpreted as a consequence of the gavage procedure and are not likely attributed to toxicological effects of the test item.
One female at 500 mg/kg bw/day (no. 83) was euthanized on Day 22 post-coitum, as she delivered her offspring early.
Description (incidence and severity):
see Table 2 - body weight and body weight changes:
Mean body weights were slightly lower in the 200 and 500 mg/kg bw/day groups compared to controls during the treatment period, but changes were not statistically significant. At the end of the treatment period, mean body weights at 200 and 500 mg/kg bw/day were 1% and 4%, respectively, lower than controls.
Mean body weight gains were up to two times lower at 200 and 500 mg/kg bw/day compared to controls, reaching statistical significance on Days 9, 15, 21 and 24 post-coitum in the 200 mg/kg bw/day group and on Days 9 to 21 post-coitum in the 500 mg/kg bw/day group.
In addition, mean body weight gains corrected for gravid uterus were slightly lower at 200 and 500 mg/kg bw/day than controls (-5.3% compared to -2.3% in the control group), but remained within the range considered normal for rabbits of this age and strain.
The marginal changes at 75 mg/kg bw/day compared to controls remained within the normal range and were not considered to be toxicologically relevant.
Description (incidence and severity):
Food consumption - treatment related effect:
Treatment at 500 mg/kg bw/day resulted in statistically significantly reduced food consumption before and after correction for body weight from Day 6 to 21 post-coitum. After correction for body weight, differences of minus 20 % (days 6-9 and 9-12 pc), minus 26% (days 12-15 pc), minus 23% (days 15-18 pc), and minus 18% (days 18-21 pc) were noted compared to controls. Thereafter, no significant changes were recorded. Remark: see also chapter 'maternal toxicity: other effects'
At 200 mg/kg bw/day, statistically significantly lower values for food consumption were noted over Days 12-15 post-coitum (absolute only) and Days 18-21 post-coitum (both absolute and relative). Over Days 18-21 post-coitum, a relative difference of 18% was noted compared to controls.
During the remaining treatment period, food consumption values of treatment groups remained in the same range as controls.
No toxicologically relevant changes were noted at 75 mg/kg bw/day.
Description (incidence and severity):
Organ weight findings:
In the 75 and 500 mg/kg bw/day groups, mean liver weight and liver/body weight ratio were higher than controls. Relative differences in ratio were 9% and 14% respectively over control, reaching statistical significance at 500 mg/kg bw/day.
The individual liver weight of female no. 70, noted with multiple liver abnormalities, remained within the normal range.
Description (incidence and severity):
Gross pathological findings:
There was an increased incidence of (dark) reddish foci on the lungs in the treatment groups compared to the control group. In total, including the macroscopic findings of the 8 premature decedents (see Table 1), lung foci were observed for 1, 8, 4 and 4 females in the control, 75, 200 and 500 mg/kg bw/day group, respectively.
One female at 500 mg/kg bw/day (animal no. 70) was noted with multiple liver abnormalities, including dark reddish discoloration and an irregular surface of the right lateral and caudate lobes and the caudate lobe grew together with the lobus dexter lateralis. As these findings were observed for a single female, they were considered to be caused by chance and of no toxicological relevance.
Description (incidence and severity):
justification for dose selection: The high dose of 500 mg/kg bw/day was considered to be the highest tolerated dose, as in the dose range finder treatment at 750 mg/kg bw/day resulted in severe toxic effects for 1/6 females, including a 9% body weight loss and absence of food consumption. Moreover, treatment at 1000 mg/kg resulted in unacceptable toxicity (i.e. body weight loss of 9-14%, absence of food consumption, piloerection and/or a lean/pale appearance) for 4/6 females followed by early euthanasia on Days 12-13 post-coitum.
Description (incidence and severity):
see Table 3 - number of abortions:
One female at 500 mg/kg bw/day (no. 83) delivered her offspring on Day 22 post-coitum. This female had a body weight loss of 5% over Days 6-21 post-coitum and a significant reduced or no food consumption over Days 15-21 post-coitum. In the animal facility, five out of ten fetuses were noted to be alive, while at necropsy all fetuses were dead. All ten fetuses were noted to be relatively small (weight was not determined). For five of them, severe external malformations were observed, including gastroschisis, omphalocele and absence of skin on the head. Remark: see also chapter 'maternal toxicity - other effects'
Description (incidence and severity):
pre and post-implantaion loss
no adverse effects observed - see Table 4: No toxicologically relevant effects on the numbers of corpora lutea and implantation sites,and pre- and post-implantation loss were noted by treatment up to 500 mg/kg bw/day.
Description (incidence and severity):
Total litter losses by resorption:
no adverse effects observed - see Tables 3 and 4
Description (incidence and severity):
Early or late resorptions:
no adverse effects observed - see Table 4
Description (incidence and severity):
see Table 4 - Dead fetuses:
Two fetuses in the 500 mg/kg bw/day group (A075-08 and A076-02) were dead at scheduled necropsy.
Description (incidence and severity):
changes in pregnancy duration:
no effects observed
Migrated Data from removed field(s)
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Effects on pregnancy duration:
no adverse effects observed; For one female at 500 mg/kg bw/day (no. 76) early delivery was noted in the morning of scheduled necropsy on Day 29 post-coitum. She delivered six pups of which one pup was dead and partly cannibalized. As no abnormalities were noted for this female, this is not considered to be toxicologically relevant.
Description (incidence and severity):
see Table 3 - changes in the number of pregnants:
There were one control female and two females each at 75, 200 and 500 mg/kg bw/day which were not pregnant. Pregnancy could not be determined for one female at 200 mg/kg bw/day (no. 53) which was moribund on Day 6 post-coitum. No implantation sites or corpora lutea were detectable for this female at necropsy, which was due to the early stage of pregnancy. This female is reported as non-pregnant in the data tables.
Description (incidence and severity):
Maternal developmental toxicity - other effects:
In one litter which was early delivered on Day 22 post-coitum, two fetuses with gastroschisis, two fetuses with omphalocele of the trunk and one fetus without skin on its head were noted and all fetuses were noted to be relatively small of size. It can be excluded that these findings were caused by cannibalism as no torn or bleeding skin layer was noted. The respective dam showed signs of toxicity; body weight loss of 5% over Days 6-21 post-coitum and a significant reduced or no food consumption over Days 15-21 post-coitum. This could indicate a delayed developmental stage of the fetuses. It is known that during embryo-fetal development of rabbits, a physiological umbilical hernia occurs which starts around gestation Day 12.5 to 14.5 and is reduced around gestation Day 20 (DeSesso, J.M. Coparative features of vertebrate embryology. Chapter 6 of Developmental reproductive toxicology: a practical approach by Taylor & Francis Group LLC, 2016). Moreover, when evaluating available historical data sets, it showed that omphalocele and/or umbilical hernia occur at an overall litter incidence of 0.3-1.1% with a lack of dose-responsiveness, and that maternal toxicity or stress (decreased food consumption and/or weight gain) may play a role in the cause of omphalocele (Daston, G., Beekhuijzen, M. Is omphalocele a non-specific malformation in New Zealand White rabbits? Reproductive Toxicology, 78, 29-39, 2018). It might be possible that at this developmental stage, mechanical forces during the early delivery process (i.e. adhesion of amniotic membranes) could have contributed to the observed wall defects. It is thus uncertain whether these malformations were related to the maternal toxicity in combination with the developmental stage of the fetuses and/or mechanical forces during the early delivery process. The early delivered fetuses were not taken into account when determining the developmental NOAEL.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: the NOAEL is the highest dose tested
Abnormalities:
no effects observed
Localisation:
other: Transient but significantly reduced body weight gain from days 9-21 pc and reduced food consumption from days 6-21 pc were recorded. Liver weight increased at 500 mg/kg bw by 14%. These observations were not considered as adverse.
Description (incidence and severity):
see Table 5 - fetal body weight changes:
Fetal body weights were lower at 500 mg/kg bw/day compared to controls. Mean combined fetal weights were 41.8, 41.3, 39.9, and 38.1 g for the control, 75, 200 and 500 mg/kg bw/day groups, respectively. At 500 mg/kg bw this weight was about 9% lower than controls. The mean values were lower than the 5th percentile of the available historical control data. However, these changes were not statistically significant and as ossification parameters were unaffected, indicating no growth retardation effects, this was considered to be non-adverse.
Male fetal weights were also lower at 75 ( and 200 mg/kg bw/day compared to the concurrent control group. The changes in these groups were considered to be caused by the relatively high control value, which was higher than the 95th percentile of the available historical control data and mainly caused by one control litter (no. A009; four male fetuses with a mean body weight of 57.6 gram). Therefore, this was not considered to be toxicologically relevant.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
see Tables 4 and 5: The numbers of alive fetuses (litters) available for fetal morphological examination were 183 (21), 134 (16), 150 (16) and 177 (19) in Groups 1, 2, 3, and 4, respectively.
Two fetuses in the 500 mg/kg bw/day group (A075-08 and A076-02) were dead at scheduled necropsy. As fetus A076-02 was partly cannibalized, no morphological examination was performed.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
see Tables 4 and 5: The male:female ratio was unaffected by treatment up to 500 mg/kg bw/day. Mean sex ratios (males:females) were 45:55, 47:53, 50:50 and 49:51 for the control, 75, 200 and 500 mg/kg bw/day groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
see Table 4: There were no treatment-related effects on litter size of any group. Mean litter sizes were 8.7, 8.4, 9.4 and 9.3 viable fetuses/litter for the control, 75, 200 and 500 mg/kg bw/day groups, respectively.
Description (incidence and severity):
see Table 6 - external malformations:
At scheduled necropsy, in one litter at 500 mg/kg bw/day one fetus (dam A068 - fetus no. 15) was found with multiple malformations, including exencephaly, gastroschisis, carpal flexure and ectrodactyly, which were, where possible, also skeletally confirmed to detect an underlying skeletal origin. During skeletal examination, sternoschisis was noted for this fetus additionally. Such a combination of malformations is not uncommon for incidentally appearing multiple malformed rabbit fetuses. Although gastroschisis was not seen previously in historical controls of the Test Facility, these malformations were regarded as an incidental finding and considered not to be related to treatment with the test item as they were observed in one single fetus at scheduled necropsy only. The other 14 fetuses in the litter did not show any abnormalities.
Other externally malformed fetuses were two control fetuses of the same litter (A015-1 and - 8) that had carpal flexures without an underlying skeletal origin and one group 2 (75 mg/kg bw) fetus (A036-8) with omphalocele. These malformations were noted previously in historical controls at comparable frequencies. As the carpal flexures observed in one litter were noted in the control group, they were not test item related. The omphalocele observed in Group 2 was regarded to be caused by chance as one single litter was affected without any other malformations noted in the low and mid dose group.
External variations were not observed in this study (see Table 7).
Description (incidence and severity):
see Table 6 - skeletal malformations:
There were no treatment related effects on skeletal morphology following treatment up to 500 mg/kg bw/day.
Skeletal malformations in fetuses of which the dam received test item were sternoschisis (Group 4 fetus A068-15 with multiple external malformations) and vertebral anomaly with associated rib anomaly (Group 4 fetuses A076-06 and A080-03).
Vertebral anomaly with or without associated rib anomaly was also observed in three fetuses of the control group (A002-04, A007-10 and A014-02) and was noted previously in historical controls. Therefore, this malformation was regarded to be unrelated to treatment.

see Table 7 - skeletal variations:
There was a statistically significantly increased litter incidence of fetuses with 13th rudimentary in Group 4 (500 mg/kg bw), compared to the concurrent control group. This variation was observed at mean litter incidences of 3.8% and 10.6% per litter in Groups 1 and 4, respectively (Groups 2 and 3 were not examined skeletally). This statistical significance was considered to have arisen as a result of a remarkably low control value, which was lower than the minimum historical control incidence of 4.8%. Moreover, the Group 4 value remained within the range of historical control data and therefore, these changes were not considered to be treatment-related.
Other skeletal variations that were noted in this study occurred at low incidences, in the absence of a dose-related incidence trend and/or at frequencies that were within the range of available historical control data.
Description (incidence and severity):
see Table 6 - visceral malformations:
There were no treatment-related effects on visceral morphology following treatment up to 500 mg/kg bw/day.
Two visceral malformations occurred in this study. Tetralogy of Fallot was observed in two fetuses from the same litter of Group 3 (A048-5 and -9) and in one control fetus (A022-2).
Internal hydrocephaly was observed in one fetus of Group 4 (A070-1) during the Wilson sectioning technique. Tetralogy of Fallot is a commonly observed finding among historical controls. Also the internal hydrocephaly was observed previously in historical control fetuses with a maximum historical litter incidence of 0.8% versus 0.5% noted in this study. In absence of a dose-related trend and as the occurrence was within the range of available historical control data, these were considered to be chance findings and not toxicologically relevant.

see Table 7 - visceral variatioins:
The total incidence of visceral variations per litter was statistically significantly increased at 500 mg/kg bw (13.4% versus 4.6% in controls, 6.8% at 75 mg/kg bw and 6.8% at 200 mg/kg bw). This was caused by an increased incidence of two variations, i.e. retrocaval ureter and absent accessory lung lobe (both not statistically significant). Mean litter incidences of retrocaval ureter were 2.1, 2.5, 2.3 and 6.0 % in Groups 1, 2, 3 and 4, respectively. In comparison to the maximum available historical control incidence of 6.3%, the increase at 500 mg/kg bw was considered not to be toxicologically relevant. In addition, the incidence of absent accessory lung lobe was increased without statistical significance in Groups 3 and 4, compared to controls. Mean litter incidences were 0.0, 1.0, 2.3 and 2.3% in Groups 1, 2, 3 and 4, respectively. As this variation was observed previously in historical control fetuses (maximum historical control value of 1.7 %) and is one of the most commonly observed variations in rabbits, these changes in Groups 3 and 4 were considered to be unrelated to treatment.
All other variations noted, were not considered treatment-related as they occurred infrequently, and/or occurred at frequencies that were within the range of the available historical control data.
Description (incidence and severity):
In this study, severe malformed fetuses were noted in one early delivered litter on Gestation Day 22 at 500 mg/kg. However, it is uncertain whether these malformations were related to the maternal toxicity in combination with the developmental stage of the fetuses and/or mechanical forces during the early delivery process. Therefore, the early delivered fetuses were not taken into account when determining the developmental NOAEL. (see also: maternal developmental toxicity - other effects)
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: the NOAEL is the highest dose tested
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1: Overview of premature decedents directly (within a few seconds to minutes) after the oral gavage treatment procedure

 Group  Animal No.  Day of necropsy  Termination  Clinical signs  Macroscopic findings
 75 mg/kg bw/day  25  day 23 post-coitum  spontaneous death  bleeding nose and labored respiration

 trachea: foamy contents

lungs: many reddish foci

   37  day 21 post-coitum  sponatneous death  clonic spasms and labored respiration

 lungs: ruptured right caudal lobe and several reddish foci

thoracic cavity: hemorrhagic / clotted blood

   38  day 07 post-coitum  euthanized in extremis  blood at the end of tube, bleeding nose and gasping  lungs: several dark reddish foci, reddish discoloration and inflation
 43  day 20 post-coitum  spontaneous death  bleeding nose and clonic spasms

 trachea: foamy contents

lungs: several dark reddish foci in the right caudal lobe

 200 mg/kg bw/day  47  day 13 post-coitum  spontaneous death  clonic spasms

 heart: hardened

trachea: foamy contents

lungs: isolated reddish foci and inflation

   53  day 06 post-coitum  euthanized in extremis

 blood at the end of tube, bleeding nose/mouth,

labored respiration, uncoordinated movements and

pale appearance

lungs: inflation  
   60  day 19 post-coitum  spontaneous death  blood at the end of tube and gasping

 lungs: perforation of the right caudal lobe, many dark reddish

foci and foamy contents

   64  day 20 post-coitum  spontaneous death  clonic spasms

 trachea: foamy contents

lungs: many dark reddish foci on the right caudal lobe

Table 2: Body weights (in g, rows 1 and 2) and body weight gain (in %, rows 3 -10) of dams

  means  control group  75 mg/kg bw/day  200 mg/kg bw/day  500 mg/kg bw/day
  day 2 pc (body weight in g)  3425  3441  3410  3386
  day 29 pc  3986  3948  3937  3819
  day 9 pc (body weight gain in %)  2  1  1*  0**
  day 12 pc  4  3  2  2*
  day 15 pc  8  5  4**  4**
  day 18 pc  8  7  5  5*
  day 21 pc  9  8  5*  5*
  day 24 pc  10  8  6*  8
  day 27 pc  11  9  8  8
  day 29 pc  12  10  9  9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 3: Summary of maternal survival and pregnancy status

  Dose group  control     75 mg/kg    200 mg/kg    500 mg/kg  
   No.  %  No.  %  No.  %  No.  %
 Females on study  22    22    22    22  
 Females that aborted or delivered  0 0.0   0  0.0  0  0.0  1  4.5
 Females that died  0 0.0  4 18.2  4  18.2  0  0.0
 Females that aborted  0  0.0  0  0.0  0  0.0  0  0.0
 Nongravid  0  0.0  0  0.0  1  25.0  0  0.0
 Gravid  0  0.0  4  100.0  3  75.5  0  0.0
 Females that were euthanized  0  0.0  0  0.0  0  0.0  0  0.0
 Nongravid  0  0.0  0  0.0  0  0.0  0  0.0
 Gravid  0  0.0  0  0.0  0  0.0  0  0.0
 Females examined at scheduled necropsy#  22  100 .0  18  81.8  18  81.8  21  95.5
 Nongravid  1  4.5  2  11.1  2  11.1  2  9.5
 Gravid  21  95.5  16  88.9  16  88.9  19  90.5

 with resorptions only

 0  0.0  0  0.0  0  0.0  0  0.0
 with viable fetuses  21  100.0  16  100.0  16  100.0  19  100.0
 Total females gravid  21  95.5  20  90.9  19  86.4  20  90.9

#- Including females that delivered Day 29: A076 (500 mg/kg bw/day)

Table 4: Summary of fetal data at scheduled necropsy

 Group  

Sex

 M

Sex 

F

 Viable

fetuses

 Dead

fetuses

Resorptions

 Early

 Resorptions

Late

 Post

implantation

loss

 Implantation

sites

 Corpora

lutea

 Pre

implantation

loss

Fetal

weights

in grams 

 No. of

gravid

females

 control  Total 83  100  183  0  4  10  14  197  212  15  NA  21
   Mean  4.0  4.8  8.7  0.0  0.2  0.5  0.7  9.4  10 .1  0.7  41.8  
 75 mg/kg  Total  65  69  134  0  6  3  9  143  149  6  NA  16
   Mean  4.1  4.3

 8.4

 0.0

 0.4

 0.2

 0.6

 8.9

 9.3

 0.4

 41.3

 

 200 mg/kg

 Total

 73

 77

 150

 0

 5

 4

 9

 159

 160

 1

 NA

 16

 

Mean 

 4.6

 4.8

 9.4

 0.0

 0.3

 0.3

 0.6

 9.9

 10.0

 0.1

 39.9

 

 500 mg/kg

 Total

 86

 91

 177

 2

 9

 2

 13

 190

 197

 7

 NA

 19

 

 Mean

 4.5

 4.8

 9.3

 0.1

 0.5

 0.1

 0.7

 10.0

 10.4

 0.4

 38.1

 

None significantly different from control group

NA = not applicable

Mean number of viable fetuses, mean number of implantation sites, mean number of corpora lutea,

Fetal weights compared using Dunnett's test

Table 5: Summary of fetal data at scheduled necropsy [% per litter]

Group 

 0 mg/kg

 75 mg/kg

 200 mg/kg

 500 mg/kg

 Number of litters

 21

 16

16

 19

Corpora lutea

 

 

 

 

 Mean

 10.1

 9.3

 10.0

 10.4

 Implantation sites

 

 

 

 

 Mean

9.4

 8.9

 9.9

 10.0

 Viable fetuses (%)

 

 

 Mean

 93.4

 93.7

 94.5

 93.4

 Dead fetuses (%)

 

 

 

 Mean

 0.0

 0.0

 0.0

 1.3

 Early resorptions (%)

 

 

 

 

 Mean

 2.8

 4.0

 3.2

 4.5

 Late resorptions (%)

 

 

 

 Mean

 3.8

 2.3

 2.2

 0.8

 Total resorptions (%)

 

 

 

 

 Mean

 6.6

 6.3

 5.5

 5.3

 Pre-implantation loss (%)

 

 

 

 

 Mean

 7.2

 3.6

 0.7

 3.6

 Post-implantation loss (%)

 

 

 

 

 Mean

 6.6

 6.3

 5.5

 6.6

 Males (%)

 

 

 

 

 Mean

 44.7

 47.4

 49.8

 49.0

 Females (%)

 

 

 

 

 Mean

 55.3

 52.6

 50.2

 51.0

 Male fetal weights (g)

 

 

 

 

 Mean

 42.6

 40.4

 39.7

 38.4

 Female fetal weights (g)

 

 

 

 

 Mean

 40.6

 41.2

 40.0

 37.6

 Combined fetal weights (g)

 

 

 

 

 Mean

 41.8

 41.3

 39.9

 38.1

Proportional (%) data compared using the Mann-Whitney test

Fetal weights compared using Dunnett's test

None significantly different from control group

Table 6: Summary of fetuses and litters with variations [absolute no.]

 

 Fetuses    

 Litters    

 Dose group (mg/kg bw per day):

 0

 75

200

500

 0

 75

 200

 500

 Number examined externally

 183

 134

 150

 177

 21

 16

 16

 19

exencephaly

 0

 0

 0

 1#

 0

0

 0

1

gastroschisis

 0

 0

 0

 1#

0

0

1

1

carpal and/or tarsal flexure

 2

 0

 0

 1#

1

 0

0

1

 omphalocele

 0

 1

 0

 0

0

1

0

0

 ectrodactyly

 0

 0

 0

 1#

0

0

0

1

Number examined viscerally

 183

 134

 150

 177

21

16

16

19

 teratology of Fallot

 1

 0

 2

 0

 1

 0

 1

 0

hydrocephaly - internal

0

 0

 0

 1

 0

0

0

1

 Number examined skeletally

 183

 0

 0

 177

 21

 0

0

 19

 vertebral anomaly with or without associated rib anomaly

 3

 -

 -

 2

 3

 -

 -

 2

 sternoschisis

 0

 -

 -

 1#

 0

 -

 -

 0

 Total number with malformations

 

 

 

 

 

 

 

 

 external

 2

 1

 0

 1

 1

 1

 0

 1

 soft tissue

 1

 0

 2

 1

 1

 0

 1

 1

 skeletal

 3

 0

 0

 3

 3

 0

0

 3

 combined  6  1  2  4  5  1  4

# fetus A068 -15 with multiple malformations

Table 7: Summary of fetuses and litters with variations

 Dose group  control  75 mg/kg

 200 mg/kg

 500 mg/kg

 

 No.

 litters

 No.

litters

 No.

 litters

 No.

 litters

 Number examined externally

 183

 21

 134

16

 150

16

177

19

 number with findings externally

 0

0

 0

0

 0

0

 0

 0

Number examined viscerally

183

21 

 134

16 

 150

16

177

19

 retrocaval ureter

 4

3

 3

3

3

1

10

8

 retrocaval ureter (mean litter incidence)

 

2.1%

 

2.5%

 

2.3%

 

6.0%

lung- absent accessory lobe

 0

0

1

1

3

2

4

3

lung- absent accessory lobe (mean litter incidence)

 

0.0%

 

1.0%

 

2.3%

2.3%

 Number examined skeletally

 183

21

-

-

 -

-

 177

19

 13th rudimentary rib(s)

 8

6

 -

 -

 -

 -

18

12

 13th rudimentary rib(s) (mean litter incidence)

 

3.8%

 -

 -

 -

 -

 

10.6%*

 Total variations

 

 

 

 

 

 

 % per litter with external variations

 

0.0

 

 0.0

 

 0.0

 

 0.0

 % per litter with soft tissue variations

4.6

 

 6.8

 

 6.8

 

13.4*

 % per litter with skeletal variations

71.6

 

 -

 

 -

 

73.4

 total % per litter with variations

 

 72.9

 

 

 

 

 

77.0

* = Significantly different from the control group at 0.05

Executive summary:

The objectives of this study were to determine the potential of the registered substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to 22 time-mated female New Zealand White rabbits per dose group from Day 6 to 28 post-coitum, inclusive. The study was performed according to OECD TG 414.

The dose levels in this study were selected to be 0, 75, 200, 500 mg/kg bw/day, based on the results of the dose range finder in which all animals of the 1000 mg/kg bw/day group and one animal of the 750 mg/kg bw/day group showed severe signs of general toxicity (e.g. severely reduced to absence of food consumption, body weight loss). The high dose of 500 mg/kg bw/day was thus considered to be the highest tolerated dose.

According to the results in the main OECD 414 prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Reaction products of phosphoryl trichloride and 2-methyloxirane (TCPP) was established as being at least 500 mg/kg bw/day, the highest dose level tested.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012: IN-LIFE DATES: From: 29 Jan 2012 (GD6) To: 15 Feb 2012 (GD20, staggered)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guideline (EPA OPPTS 870.3700) and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Citation: Organisation for Economic Co-operation and Development (OECD). OECD guideline for the testing of chemicals: Proposal for updating guideline 414. Prenatal developmental toxicity study. Paris, France: Organisation for Economic Co-operation and Development; 2001.
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Citation: U.S. Environmental Protection Agency (USEPA). Guidelines for developmental toxicity risk assessment. Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum; 1991. EPA Document No. EPA/600/FR-91/001.
Principles of method if other than guideline:
Both the EPA EPA OPPTS 870:3700 and OECD 414 studies were listed in study references. Although neither was declared as being the primary test guideline in use, the study appears to follow the principles outlined within the EPA OPPTS 870:3700 study closely.
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Hsd:Sprague Dawley® SD®
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo (formerly Harlan Laboratories, Inc., Dublin, VA)
- Age at study initiation: 12 to 13 weeks
- Weight at study initiation: 203.5 to 256.0 g on GD 3
- Fasting period before study: N/A
- Housing: Solid-bottom polycarbonate cages (Lab Products, Inc., Seaford, DE), changed and rotated weekly
- Diet (e.g. ad libitum): Irradiated NIH-07 Certified Rodent Diet wafer diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum
- Water (e.g. ad libitum): Tap water (Durham, NC, municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum
- Acclimation period: N/A - time mated.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72°F ± 3°F (22˚C)
- Humidity (%): 50% ± 15%
- Air changes (per hr): at least 10/h
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Jan 2012 (GD6) To: 15 Feb 2012 (GD20, staggered)
Route of administration:
oral: gavage
Vehicle:
other: methylcellulose 0.5%
Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Methylcellulose was used in previous studies (dose range finder)
- Concentration in vehicle: 0.5%
- Amount of vehicle (if gavage): amount required to produce dosing volume 5 mL/kg when mixed with TCPP
- Lot/batch no. (if required): 2AJ0439
- Purity: Methoxy group content was 31.0% - within the accepted range of 27.5–31.5%.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the prenatal developmental toxicity study, the dose formulations were prepared three times and analyzed once via GC/FID; all nine dose formulation samples were within 10% of the target concentrations.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
GD6-20
Frequency of treatment:
1x Daily
Duration of test:
GD 1/2 (receipt of animal) - GD 21 (necropsy)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
in 0.5% methylcellulose. Dosing volume 5 mL/kg
Dose / conc.:
162.5 mg/kg bw/day (nominal)
Remarks:
in 0.5% methylcellulose. Dosing volume 5 mL/kg
Dose / conc.:
325 mg/kg bw/day (nominal)
Remarks:
in 0.5% methylcellulose. Dosing volume 5 mL/kg
Dose / conc.:
650 mg/kg bw/day (nominal)
Remarks:
in 0.5% methylcellulose. Dosing volume 5 mL/kg
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on preliminary dose range finding study.
- Rationale for animal assignment (if not random): Random
- Fasting period before blood sampling for (rat) dam thyroid hormones: Thyroid hormones not examined.
- Time of day for (rat) dam blood sampling: not specified
- Other: N/A
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 2x daily GD3-20

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 1x daily from GD3 - necropsy (GD21)

BODY WEIGHT: Yes
- Time schedule for examinations: 1x daily

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: N/A

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: terminal body, adrenal glands, liver, ovary, and gravid uterine weights were recorded and the uterine contents examined.

OTHER: N/A
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number and location of all fetuses (live or dead)
Blood sampling:
- Plasma: No
- Serum: No
- Volume collected: N/A
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]
- Anogenital distance of all live rodent pups: No
Statistics:
Statistical analyses were performed on data from pregnant females that survived until the end of the study and were examined on GD 21 and from live fetuses. Statistical analyses were performed using SAS 9.3 (SAS Institute, Cary NC) software.
Maternal parameteres: continuous variables (body weight, feed consumption, organ weights etc) summarised with mean and SE. Placental and Fetal parameters: mean and SE of litter means, % of live fetuses, Incidence reported as % of affected compared to total.
Maternal organ/bodyweight: w. normal distribution = parametric Dunnett and Wiliams / w. non-normally distributed data = Shirley & Dunn multiple comparison. Jonckheere test was used to assess the significance of dose-related trends. Prior to statistical analysis, extreme values identified by the Dixon and Massey outlier test were examined and implausible values eliminated from the analysis. Fetal body weights were analyzed using mixed-effects linear models, with litter as a random effect to account for potential within-litter correlations. For pairwise comparisons with the control group, a second mixed-effects model with dose entered into the model as a categorical variable was estimated, followed by the Dunnett and Hsu multiple comparison tests.

Historical control data:
NTP historical controls are available online at https://ntp.niehs.nih.gov/go/historical_controls
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Dose-related clinical observations were observed in six females in the 650 mg/kg group and included nasal discharge, salivation, twitches, ataxia, piloerection, audible respiratory sounds, and hyperactivity. The duration for most of the clinical observations was limited to 1 day of gestation aside from hyperactivity in one female, which was observed over a 7-day period starting on GD 7. No dose-related clinical effects were observed in the other TCPP groups or in the vehicle control (0 mg/kg) animals.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Maternal disposition is summarised in table 01.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No dose-related effects on maternal body weight gain during gestation were found in any dose group. Table 02.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Compared to the vehicle control group, maternal feed consumption was 8–16% lower over GD 6 to GD 9 and GD 9 to GD 12 for dams in the 650 mg/kg group. The feed consumption differences were transient, and, overall, TCPP administration had no effect on maternal feed consumption during gestation. Table 03.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Dose-related increases in absolute (9%, 16%, and 26% at 162.5, 325, and 650 mg/kg, respectively) and relative liver weights (37.93, 40.78, 43.39 and 48.09 mg organ/g bodyweight at 0, 162.5, 325, and 650 mg/kg, respectively). Table 04.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no maternal gross observations at necropsy
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Although a twofold increase in mean percent postimplantation loss in the 650 mg/kg group as compared to the vehicle controls was found, this increase is the result of one dam that had nine early resorptions. Given the singular litter incidence, this finding in the 650 mg/kg group was not considered related to TCPP administration.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Uterine content data is summarised within table 05.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 650 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No exposure-related effects on absolute fetal body weights (male or female) were evident. Table 05.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
The number of live fetuses per litter was 5% lower in the 325 and 650 mg/kg groups and was accompanied by lower gravid uterine weights (<7%) at these doses; however, these differences were not statistically significant. Table 05.
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the external exam, various low or single-incidence findings were observed in the head and placenta across all groups that were considered unrelated to exposure.

The only malformation, a meningoencephalocele in the 162.5 mg/kg group, was also considered unrelated to TCPP exposure because of the single incidence and lack of a dose response. Table 06.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Single incidences of malformations occurred in the head soft tissue including enlarged nasal sinus, anophthalmia, and folded retina that were noted in vehicle control animals and groups exposed to TCPP. The only variation, enlarged lateral ventricle of the brain, was observed in the same fetus with anophthalmia and meningoencephalocele in the 162.5 mg/kg group. Overall, there were no effects of TCPP exposure on the incidences of fetal head abnormalities.
Skeletal malformations including discontinuous rib cartilage and full lumbar ribs were observed in TCPP-exposed animals. The incidences of these findings were low, however, and considered not exposure related. Associated skeletal variations observed in TCPP treated groups included incomplete ossification of the sternebrae (II and V), floating extra rib, rudimentary ribs (lumbar I), and bipartite or dumbbell ossification of the thoracic centrum. A statistically significant increase (trend and pairwise comparison) was found for the percentage of fetuses with rudimentary ribs in all TCPP groups (22%, 23%, 22%) compared to the concurrent controls (14%). Although this finding appears to be exposure related, it is not dose dependent. This lack of dose response, a variation of limited biological significance, and lack of any other related effect suggests that the finding is not toxicologically relevant. Overall, examination of the fetal skeleton for osseous and cartilaginous defects of the skull (~50% of fetuses) and body only (100% of the fetuses) was not suggestive of an effect related to TCPP. Table 07.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations and associated variations were observed in the ureter, which included hydroureter malformations (bilateral and unilateral) in the vehicle control and 325 mg/kg groups and distention of the ureter (bilateral and unilateral) across all groups. Although associated, these findings were not considered exposure related because the incidences of findings in groups exposed to TCPP were either similar to or lower than the incidences in vehicle control animals. Various other single-incidence malformations and variations were observed in the abdomen, heart, and thorax following the visceral exam; these findings were either not considered exposure related or observed only in vehicle control animals. Overall, there were no effects of TCPP exposure on the incidences of fetal visceral abnormalities
Details on embryotoxic / teratogenic effects:
Fetal effects are summarised within tables 06 (visceral) and 07 (skeletal).
Key result
Dose descriptor:
NOAEL
Effect level:
>= 650 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Supplemental data including summary and individual data tables is available within Appendix E of the NTP study report: https://pubmed.ncbi.nlm.nih.gov/32716615/ 


 


Table 01: Maternal Disposition of Rats in the Prenatal Developmental Toxicity Gavage Study of Tris(chloropropyl) Phosphate


































 



0 mg/kga



162.5 mg/kg



325 mg/kg



650 mg/kg



Time-mated Females



50



25



25



25



 Pregnant (on GD 21)



44



21



21



20



 Nonpregnant (on GD 21)



6



4



4



5



GD = gestation day. aThis study had two vehicle control groups. Data from both vehicle control groups were combined and are presented here.


 


Table 02. Summary of Maternal Body Weight Gains of Rats in the Prenatal Developmental Toxicity Gavage Study of Tris(chloropropyl) Phosphate 






























































Gestation Day Interval



0 mg/kg



162.5 mg/kg



325 mg/kg



650 mg/kg



6–21



139.6 ± 2.5a (44)



143.6 ± 3.0 (21)



139.9 ± 3.8 (21)



137.6 ± 6.3 (20)



3–6



14.4 ± 0.8 (44)



14.1 ± 0.7 (21)



15.7 ± 1.0 (21)



14.1 ± 1.0 (20)



6–9



12.9 ± 0.6 (44)



13.0 ± 0.6 (21)



12.5 ± 0.8 (21)



10.6 ± 0.8 (20)



9–12



16.8 ± 0.6 (44)



15.5 ± 1.1 (21)



16.8 ± 0.8 (21)



15.4 ± 1.2 (20)



12–15



18.9 ± 0.9 (44)



20.5 ± 0.9 (21)



19.6 ± 1.4 (21)



21.2 ± 1.3 (20)



15–18



42.4 ± 1.0 (44)



43.7 ± 1.0 (21)



41.9 ± 1.6 (21)



39.0 ± 2.2 (20)



18–21



48.8 ± 1.4 (44)



50.8 ± 1.5 (21)



49.0 ± 1.8 (21)



51.4 ± 3.1 (20)



Statistical analysis performed by the Jonckheere test (trend) or the Williams or Dunnett test (pairwise comparison) found no statistically significant trend or pairwise comparison.


aBody weight gains for pregnant females are given in grams. Data are displayed as mean ± standard error. Number of dams weighed is given in parentheses.        


 


Table 03. Summary of Maternal Feed Consumption of Rats in the Prenatal Developmental Toxicity Gavage Study of Tris(chloropropyl) Phosphate
































































Gestation Day Interval0 mg/kg162.5 mg/kg325 mg/kg650 mg/kg
6–2122.8 ± 0.23a (44)22.5 ± 0.33 (21)22.9 ± 0.33 (21)22.2 ± 0.42 (20)
3–620.2 ± 0.42 (44)18.7 ± 0.24 (21)19.7 ± 0.50 (21)19.4 ± 0.55 (20)
6–920.4 ± 0.32** (44)19.4 ± 0.37 (21)18.8 ± 0.41** (21)17.0 ± 0.46** (20)
9–1221.6 ± 0.34** (44)20.7 ± 0.41 (21)21.0 ± 0.38 (21)19.8 ± 0.54** (20)
12–1521.6 ± 0.22 (44)21.1 ± 0.37 (21)22.4 ± 0.55 (21)21.4 ± 0.29 (20)
15–1824.8 ± 0.34 (44)25.0 ± 0.42 (21)25.5 ± 0.53 (21)25.2 ± 0.74 (20)
18–2125.5 ± 0.36** (44)26.3 ± 0.51 (21)27.0 ± 0.38* (21)27.8 ± 0.66** (20)






 



*Statistically significant (p 0.05) trend (by the Jonckheere test) or pairwise comparison (by the Shirley or Dunn test). A significant trend test is indicated in the vehicle control column. A significant pairwise comparison with the vehicle control group is indicated in the dose group column. 


**p 0.01.


aFeed consumption for pregnant females is given in grams per day. Data are displayed as mean ± standard error. Number of dams with feed consumption measured is given in parentheses.


 


Table 04. Summary of Maternal Liver Weights and Liver Weight Ratios for Rats in the Prenatal Developmental Toxicity Gavage Study of Tris(chloropropyl) Phosphatea 









































 



0 mg/kg



162.5 mg/kg



325 mg/kg



650 mg/kg



n



44



21



21



20



Necropsy Body Wt.



378.5 ± 3.0



382.7 ± 4.0



383.1 ± 5.2



375.2 ± 8.3



Liver


 Absolute 



 


14.35 ± 0.19**



 


15.62 ± 0.32**



 


16.58 ± 0.27**



 


18.02 ± 0.56**



 Relative 



37.93 ± 0.41**



40.78 ± 0.67**



43.39 ± 0.78**



48.09 ± 1.09**



**Statistically significant (p 0.01) trend (by the Jonckheere test) or pairwise comparison (by the Williams or Dunnett test). A significant trend test is indicated in the vehicle control column. A significant pairwise comparison with the vehicle control group is indicated in the dose group column.


aLiver weights (absolute weights) and body weights are given in grams; liver weight-to-body weight ratios (relative weights) are given as mg organ weight/g body weight. Data are displayed as mean ± standard error.


 


Table 05. Summary of Uterine Content Data for Rats in the Prenatal Developmental Toxicity Gavage Study of Tris(chloropropyl) Phosphate 

























































































































































































 



0 mg/kg



162.5 mg/kg



325 mg/kg



650 mg/kg



Pregnancy Summary                        


 Mated females



 


50



 


25



 


25



25



 Pregnant females



44



21



21



20



 Pregnant females examined on   GD 21a



44



21



21



20



 Corpora lutea per female



16.64 ± 0.37 (44)



18.05 ± 0.55 (21)



16.62 ± 0.72 (21)



17.55 ± 0.85 (20)



 Implantations per female



14.18 ± 0.29 (44)



14.81 ± 0.38 (21)



13.48 ± 0.65 (21)



13.70 ± 0.67 (20)



 Percent postimplantation lossb



3.81 ± 1.13 (44)



3.42 ± 0.99 (21)



4.33 ± 1.19 (21)



7.17 ± 4.50 (20)



 Total resorptions per litterb



0.55 ± 0.16 (44)



0.52 ± 0.15 (21)



0.52 ± 0.15 (21)



0.75 ± 0.45 (20)



 Early resorptions per litterb



0.50 ± 0.16 (44)



0.52 ± 0.15 (21)



0.52 ± 0.15 (21)



0.75 ± 0.45 (20)



 Late resorptions per litterb



0.05 ± 0.03 (44)



0.00 ± 0.00 (21)



0.00 ± 0.00 (21)



0.00 ± 0.00 (20)



 Dead fetuses per litterb



0.02 ± 0.02 (44)



0.00 ± 0.00 (21)



0.10 ± 0.07 (21)



0.00 ± 0.00 (20)



 Number of early resorptionsc



22



11



11



15



 Number of late resorptionsc



2



0



0



0



 Number of whole litter   resorptionsa



0



0



0



0



 Number of dead fetusesc



1



0



2



0



Live Fetusesb                                 


 Number of live fetuses



 


599



 


300



 


270



259



 Live fetuses per litter



13.61 ± 0.30 (44)



14.29 ± 0.37 (21)



12.86 ± 0.62 (21)



12.95 ± 0.91 (20)



 Live male fetuses per litter



6.48 ± 0.28 (44)



7.57 ± 0.41 (21)



6.71 ± 0.54 (21)



6.60 ± 0.54 (20)



 Live female fetuses per litter



7.14 ± 0.30 (44)



6.71 ± 0.52 (21)



6.10 ± 0.46 (21)



6.35 ± 0.66 (20)



 Percent live male fetuses   per litter



47.71 ± 1.88 (44)



53.48 ± 3.05 (21)



50.51 ± 3.79 (21)



50.04 ± 3.96 (20)



Fetal Weight (g)d                            


 Fetal weight per litter



 


5.29 ± 0.04 (44)



 


5.22 ± 0.06 (21)



 


5.42 ± 0.08 (21)



5.22 ± 0.07 (20)



 Male fetal weight per litter



5.42 ± 0.05 (44)



5.35 ± 0.06 (21)



5.47 ± 0.06 (20)



5.34 ± 0.06 (19)



 Female fetal weight per litter



5.17 ± 0.04 (44)



5.08 ± 0.06 (21)



5.30 ± 0.09 (21)



5.09 ± 0.07 (20)



Gravid Uterine Weight (g)e               


 Gravid uterine weight



 


98.89 ± 1.93 (44)



 


101.98 ± 2.23 (21)



 


95.76 ± 4.06 (21)



91.78 ± 5.91 (20)



 Terminal body weight



378.5 ± 3.0 (44)



382.7 ± 4.0 (21)



383.1 ± 5.2 (21)



375.2 ± 8.3 (20)



 Adjusted body weight



279.56 ± 1.81 (44) 280.75 ± 2.23 (21) 287.30 ± 2.38 (21) 283.45 ± 3.85 (20)



Values are reported per litter as mean ± standard error (n) and do not include nonpregnant females or those that did not survive to end of study. GD = gestation day.


aStatistical analysis performed by the Cochran-Armitage (trend) and Fisher exact (pairwise) tests.


bStatistical analysis performed by the Jonckheere (trend) and Shirley or Dunn (pairwise) tests.


cNo statistical analyses were performed on the number of early resorptions, number of late resorptions, or number of dead fetuses.


dStatistical analysis performed using a mixed-effects linear model with litter as a random effect (trend and pairwise).


eStatistical analysis performed by the Jonckheere (trend) and Williams or Dunnett (pairwise) tests; adjusted body weight = terminal body weight minus gravid uterine weight.


 


 


Table 06. Summary of Selected Fetal Visceral Findings in Rats in the Prenatal Developmental Toxicity Gavage Study of Tris(chloropropyl) Phosphate





















































































































































 



0 mg/kg



162.5 mg/kg



325 mg/kg



650 mg/kg



Total Number of Fetuses 



599



300



270



259



Number of Fetuses Examined



599



299



270



259



Number of Litters Examined



44



21



21



20



Pelvis


 Ureter


  Total, hydroureter – [M]


   Fetuses



 


 


 


1 (0.17)



 


 


 


0 (0.00)



 


 


 


1 (0.37)



 


 


 


0 (0.00)



   Litters



1 (2.27)



0 (0.00)



1 (4.76)



0 (0.00)



  Bilateral, hydroureter – [M]


   Fetuses



 


1 (0.17)



 


0 (0.00)



 


0 (0.00)



 


0 (0.00)



   Litters



1 (2.27)



0 (0.00)



0 (0.00)



0 (0.00)



   Left, hydroureter – [M]


   Fetuses



 


0 (0.00)



 


0 (0.00)



 


1 (0.37)



 


0 (0.00)



   Litters



0 (0.00)



0 (0.00)



1 (4.76)



0 (0.00)



  Total, distended – [V]


   Fetuses



 


92 (15.36)**#



 


35 (11.71)



 


14 (5.19)**##



 


24 (9.27)**



   Litters



30 (68.18)



15 (71.43)



5 (23.81)**



12 (60.00)



  Bilateral, distended – [V]


   Fetuses



 


47 (7.85)**#



 


14 (4.68)*



 


7 (2.59)**#



 


10 (3.86)*



 



0 mg/kg



162.5 mg/kg



325 mg/kg



650 mg/kg



   Litters



19 (43.18)



10 (47.62)



3 (14.29)*



6 (30.00)



 Left, distended – [V]


   Fetuses



 


22 (3.67)



 


9 (3.01)



 


4 (1.48)



 


9 (3.47)



   Litters



16 (36.36)



8 (38.10)



3 (14.29)



7 (35.00)



 Right, distended – [V]


   Fetuses



 


23 (3.84)*



 


12 (4.01)



 


3 (1.11)*#



 


5 (1.93)



   Litters



15 (34.09)



4 (19.05)



1 (4.76)**



4 (20.00)


        

Upper row denotes number of affected fetuses and (%) and lower row the number of affected litters and (%).


Statistical analysis for litter data and for fetal data (without the litter effects) performed by the Cochran-Armitage (trend) and Fisher exact (pairwise) tests.


*Statistically significant (p 0.05) trend (denoted in vehicle control column) or pairwise comparison (denoted in dose group column).  **p 0.01.


Statistical analysis of fetuses with litter-based adjustments performed by mixed-effects logistic regression.


#Statistically significant (p 0.05) trend (denoted in vehicle control column) or pairwise comparison (denoted in dose group column) in litter-based analysis of fetuses. 


##p 0.01.


 


Table 07. Summary of Selected Fetal Skeletal Findings in Rats in the Prenatal Developmental Toxicity Gavage Study of Tris(chloropropyl) Phosphate









































































































































































































































































































































                                                                    0 mg/kg



162.5 mg/kg



325 mg/kg



650 mg/kg



Total Number of Fetuses                                     599



300



270



259



Number of Fetuses Examined                               599



300



270



259



Number of Litters Examined                                 44



21



21



20



Thoracic Vertebrae – Thoracic Centrum


 Fused – [M]                                                        


  Fetuses                                                        1 (0.17)



 


0 (0.00)



 


0 (0.00)



 


0 (0.00)



  Litters                                                         1 (2.27)



0 (0.00)



0 (0.00)



0 (0.00)



 Bipartite ossification, bipartite cartilage – [M]


  Fetuses                                                        1 (0.17)



0 (0.00)



0 (0.00)



0 (0.00)



  Litters                                                         1 (2.27)



0 (0.00)



0 (0.00)



0 (0.00)



 Unilateral ossification, bipartite cartilage – [M]


  Fetuses                                                        1 (0.17)



0 (0.00)



0 (0.00)



0 (0.00)



  Litters                                                         1 (2.27)



0 (0.00)



0 (0.00)



0 (0.00)



 Unossified, bipartite cartilage – [M]                         


  Fetuses                                                        1 (0.17)



 


0 (0.00)



 


0 (0.00)



 


0 (0.00)



  Litters                                                         1 (2.27)



0 (0.00)



0 (0.00)



0 (0.00)



 Bipartite ossification, normal cartilage – [V] 


  Fetuses                                                        2 (0.33)



3 (1.00)



1 (0.37)



1 (0.39)



  Litters                                                         2 (4.55)



2 (9.52)



1 (4.76)



1 (5.00)



 Bipartite ossification, dumbbell cartilage – [V]


  Fetuses                                                        1 (0.17)



0 (0.00)



2 (0.74)



1 (0.39)



  Litters                                                         1 (2.27)



0 (0.00)



2 (9.52)



1 (5.00)



 Bipartite ossification, normal or dumbbell cartilage – [V]


  Fetuses                                                        3 (0.50)



3 (1.00)



3 (1.11)



2 (0.77)



  Litters                                                         3 (6.82)



2 (9.52)



3 (14.29)



1 (5.00)



 Dumbbell ossification, normal cartilage – [V]


  Fetuses                                                        7 (1.17)



4 (1.33)



4 (1.48)



7 (2.70)



  Litters                                                        6 (13.64)



3 (14.29)



4 (19.05)



3 (15.00)



 Dumbbell ossification, dumbbell cartilage – [V]


  Fetuses                                                        3 (0.50)



1 (0.33)



2 (0.74)



2 (0.77)



  Litters                                                         2 (4.55)



1 (4.76)



2 (9.52)



2 (10.00)



 Dumbbell ossification, normal or dumbbell cartilage – [V]


  Fetuses                                                       10 (1.67)



5 (1.67)



6 (2.22)



9 (3.47)



  Litters                                                        8 (18.18)



4 (19.05)



5 (23.81)



4 (20.00)



 Cartilage, normal ossification, dumbbell cartilage – [V]


  Fetuses                                                        0 (0.00)



1 (0.33)



0 (0.00)



0 (0.00)



  Litters                                                         0 (0.00)



1 (4.76)



0 (0.00)



0 (0.00)



Ribs                                                                    


 Cartilage, discontinuous – [M]                                


  Fetuses                                                        0 (0.00)



 


 


1 (0.33)



 


 


0 (0.00)



 


 


0 (0.00)



  Litters                                                         0 (0.00)



1 (4.76)



0 (0.00)



0 (0.00)



 



0 mg/kg



162.5 mg/kg



325 mg/kg



650 mg/kg



 Cartilage, VIII attached to sternum – [M]   Fetuses



 


1 (0.17)



 


0 (0.00)



 


0 (0.00)



 


0 (0.00)



  Litters



1 (2.27)



0 (0.00)



0 (0.00)



0 (0.00)



 Discontinuous – [M]   Fetuses



 


1 (0.17)



 


0 (0.00)



 


0 (0.00)



 


0 (0.00)



  Litters



1 (2.27)



0 (0.00)



0 (0.00)



0 (0.00)



 Floating extra – [V]   Fetuses



 


0 (0.00)



 


0 (0.00)



 


1 (0.37)



 


0 (0.00)



  Litters



0 (0.00)



0 (0.00)



1 (4.76)



0 (0.00)



 Left, intercostal rib – [M]


  Fetuses



 


1 (0.17)



 


0 (0.00)



 


0 (0.00)



 


0 (0.00)



  Litters



1 (2.27)



0 (0.00)



0 (0.00)



0 (0.00)



 Lumbar I full – [M]   Fetuses



 


4 (0.67)



 


4 (1.33)



 


2 (0.74)



 


3 (1.16)



  Litters



4 (9.09)



3 (14.29)



1 (4.76)



2 (10.00)



 Lumbar I rudimentarya – [V]   Fetuses



 


82 (13.69)**#



 


65 (21.67)**



 


61 (22.59)**#



 


56 (21.62)**



  Litters



29 (65.91)



17 (80.95)



17 (80.95)



15 (75.00)



Sternebrae


 Sternebra II, incomplete ossification – [V]   Fetuses



 


1 (0.17)



 


0 (0.00)



 


0 (0.00)



 


0 (0.00)



  Litters



1 (2.27)



0 (0.00)



0 (0.00)



0 (0.00)



 Sternebra V, incomplete ossification —[V]   Fetuses



2 (0.33)



1 (0.33)



4 (1.48)



0 (0.00)



  Litters



2 (4.55)



1 (4.76)



3 (14.29)



0 (0.00)



 Sternebra(e), extra ossification site between sternebrae – [V]


  Fetuses                                                        3 (0.50)



1 (0.33)



0 (0.00)



0 (0.00)



  Litters                                                         2 (4.55)



1 (4.76)



0 (0.00)



0 (0.00)



 Sternebra(e), Total, Incomplete Ossification – [V]                  


  Fetuses                                                        3 (0.50)



 


1 (0.33)



 


4 (1.48)



0 (0.00)



  Litters                                                         3 (6.82)



1 (4.76)



3 (14.29)



0 (0.00)



 Sternebra(e), misaligned (>2, not V) – [V]


  Fetuses                                                        1 (0.17)



0 (0.00)



0 (0.00)



0 (0.00)



  Litters                                                         1 (2.27)



0 (0.00)



0 (0.00)



0 (0.00)



Upper row denotes number of affected fetuses and (%) and lower row the number of affected litters and (%).


Statistical analysis for litter data and for fetal data (without the litter effects) performed by the Cochran-Armitage (trend) and Fisher exact (pairwise) tests. 


**Statistically significant (p 0.01) trend (denoted in vehicle control column) or pairwise comparison (denoted in dose group column) in litter-based analysis of fetuses.


Statistical analysis of fetuses with litter-based adjustments performed by mixed-effects logistic regression.


#Statistically significant (p 0.05) trend (denoted in vehicle control column) or pairwise comparison (denoted in dose group column) in litter-based analysis of fetuses.


[M] = malformation; [V] = variation.


aHistorical incidence for all routes: Fetuses 114/1,385 (8.23%), range 3.35–13.69%; Litters 53/97 (54.64%), range 26.32–


65.91%.                                                                         


[M] = malformation; [V] = variation.

Conclusions:
Under the conditions of this prenatal study, no evidence of developmental toxicity of TCPP was found in Hsd:Sprague Dawley® SD® rats administered 162.5, 325, or 650 mg/kg/day in the absence of overt maternal toxicity. In conclusion, based on the results of this EPA OPPTS 870.3700 prenatal developmental toxicity study, the following No Observed Adverse Effect Level (NOAEL) of Reaction products of phosphoryl trichloride and 2-methyloxirane (TCPP) was established:

Developmental NOAEL: at least 650 mg/kg/day (maternal and fetal)
Executive summary:

The purpose of this report is to summarize and discuss TCPP effects on prenatal development. In this study, time-mated female Sprague Dawley (Hsd:Sprague Dawley® SD®) rats received TCPP (95.7–97% pure) in 0.5% methylcellulose by gavage from implantation on gestation day (GD) 6 to the day before expected parturition (GD 20). Evidence of TCPP-related maternal and fetal toxicity was examined in a guideline prenatal developmental toxicity study. 


 


 


 


Groups of 25 time-mated female rats were administered 0 (2 concurrent vehicle control groups), 162.5, 325, or 650 mg TCPP/kg body weight (based on the most recent weight) per day in 0.5% aqueous methylcellulose by gavage from GD 6 to GD 20 (15 days). The additional vehicle control group was included to generate additional control data for both maternal and fetal findings in this strain of rat. At the end of the study, the vehicle control groups were evaluated for reproducibility and combined for assessment of treatment-related effects because they were run concurrently. Vehicle control animals received aqueous methylcellulose alone; the dosing volume was 5 mL/kg.


 


On GD 21, fetuses were removed from the uterus, and live fetuses individually weighed. The uteri of animals that did not appear pregnant were examined for nidations (implantation sites) by staining with 0.5% ammonium sulfide. All fetuses were examined externally for alterations, including inspection of the oral cavity for cleft palate. Live fetuses were subsequently euthanized by intraperitoneal injection of sodium pentobarbital. Fetal sex was confirmed by inspection of gonads in situ. All fetuses were examined for soft tissue alterations under a stereomicroscope. The heads were removed from approximately half the fetuses in each litter and fixed in Bouin’s solution and subsequently examined by free-hand sectioning. This technique precludes skeletal evaluations of the skull; therefore, remaining heads and all fetuses were eviscerated, fixed in ethanol, macerated in potassium hydroxide, stained with alcian blue and alizarin red, and examined for subsequent cartilage and osseous alterations. External, visceral, and skeletal fetal alterations were recorded as developmental variations or malformations. 


 


Examination of fetuses for malformations or variations in the prenatal developmental toxicity study demonstrated that TCPP exposure does not cause toxicologically significant external, visceral, or skeletal defects. The only statistically significant exposure-related finding was an increase in the percentage of fetuses with lumbar rudimentary ribs. In NTP’s experience, this skeletal variation is variable among this strain of rat. Rudimentary ribs have been reported to be common in laboratory rodents and are considered reversible and of limited toxicological relevance.


 


Under the conditions of this prenatal study, no evidence of developmental toxicity of TCPP was found in Hsd:Sprague Dawley® SD® rats administered 162.5, 325, or 650 mg/kg/day in the absence of overt maternal toxicity.               

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The endpoint is concluded based on a single study assigned a Klimisch rating of 1: reliable without restriction. This has been undertaken according to GLP and an accepted OECD TG for this endpoint (TG414). The database for this endpoint met all relevant data requirements under REACH for the respective tonnage band.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Short description of key information: 


 


A weight of evidence (WoE) approach is used for developmental toxicity, by means of studies in the rat and rabbit performed with the test substance using oral (gavage) dosing. These are outlined in brief below.


 


Key study: Oral | Rat | OECD 414 | NTP | 2020


Groups of 25 time-mated female rats were administered 0 (2 concurrent vehicle control groups), 162.5, 325, or 650 mg TCPP/kg body weight (based on the most recent weight) per day in 0.5% aqueous methylcellulose by gavage from GD 6 to GD 20 (15 days). On Gestation Day 21 the dams were sacrificed, and the foetuses were examined for external, visceral, and skeletal malformations and variations.


 


Results


Examination of fetuses for malformations or variations in the prenatal developmental toxicity study demonstrated that TCPP exposure does not cause toxicologically significant external, visceral, or skeletal defects. The only statistically significant exposure-related finding was an increase in the percentage of fetuses with lumbar rudimentary ribs. In NTP’s experience, this skeletal variation is variable among this strain of rat. Rudimentary ribs have been reported to be common in laboratory rodents and are considered reversible and of limited toxicological relevance.


 


Conclusions


Under the conditions of this prenatal study, no evidence of developmental toxicity of TCPP was found in Hsd:Sprague Dawley® SD® rats administered 162.5, 325, or 650 mg/kg/day in the absence of overt maternal toxicity.  


The maternal NOAEL was ≥ 650 mg/kg bw/day


The NOAEL for developmental effects was ≥ 650 mg/kg bw/day


 


 


Key study: Oral | Rabbit | OECD 414 | van de Ven | 2018


Following the ECHA Final Decision on a Compliance Check, Decision No. CCH-D-2114344289 -42 -01/F, a pre-natal developmental toxicity study (OECD 414) on rabbits was performed. 22 time-mated female New Zealand White rabbits per group were treated orally by gavage from Day 6 to 28 post-coitum, inclusive. The dose levels in this study were selected to be 0, 75, 200, 500 mg/kg bw/day, based on the results of the dose range finder in which all animals of the 1000 mg/kg bw/day group and one animal of the 750 mg/kg bw/day group showed severe signs of general toxicity (e.g. severely reduced to absence of food consumption, body weight loss).


 


 


Results


According to the results in the main OECD 414 prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Reaction products of phosphoryl trichloride and 2-methyloxirane (TCPP) was established as being at least 500 mg/kg bw/day, the highest dose level tested. 


 


Conclusions


Under the conditions of this prenatal study, no evidence of developmental toxicity was found in New Zealand White rabbits exposed to 0, 75, 200, 500 mg/kg bw/day TCPP:


The maternal NOAEL was ≥ 500 mg/kg bw/day


The NOAEL for developmental effects was ≥ 500 mg/kg bw/day


 


WoE study: Oral | Rat | non guideline / GLP pre- and post-natal developmental study| Kawasaki et al | 1982


Pregnant Wistar rats were administered TCPP in solid food from days 0 to 20 of gestation in doses of 1% (about 510 mg/kg bw/day), 0.1% (about 54 mg/kg bw/day) and 0.01% (about 5 mg/kg bw/day).  In the first phase groups, foetuses were necropsied on day 20 of gestation and examined for skeletal and visceral abnormalities. In the post-natal phase, dams were given 0.01 (7 dams), 0.1 (6 dams) and 1% (5 dams) TCPP in the diet up to weaning.  Pups were weaned 21 days after birth and monitored until 4 weeks. The litters of dams fed TCPP in the diet throughout pregnancy were adjusted to an average of 8 newborns each within each group and were reared for three weeks with the dams.


 


Results


At term, maternal body weight gain and food intake in all examined groups were almost equal to the control. There were no differences among all the groups in the numbers of implantations, resorptions and fetal body weight. No increased incidence of gross, skeletal or visceral fetal malformations were identified in any groups when compared to control. Development of neonates in all groups (5 -7 litters per group) were equal to the control.


Conclusions


Under the conditions tested, it was concluded that TCPP possesses no teratogenic effect in rats fed the diet containing ranging from 0.01 to 1% during the whole period of gestation. The NOEL for developmental toxicity was found to be 571 mg/kg bw.


 


 


 


WoE study: Oral | Rat | OECD 416 2 generation reproduction toxicity study | Waalkens-Berendsen | TNO | 2007


In a Two-Generation Reproduction Toxicity Study according OECD Guideline 416, 28 Wistar rats received diets containing 0, low-, mid- and high-dose levels of TCPP (84.6 - 988.2 mg/kg bw) over two successive generations. The animals were exposed during the premating period of at least 10 weeks, during gestation and lactation until sacrifice. Dams were allowed to raise one litter. At the end of the lactation period pups were weaned and selected for the next generation or sacrificed. F0- and F1- dams were sacrificed at or shortly after weaning. F0 -and F1 -males were sacrificed after at least 11 weeks of exposure for sperm analyses and necropsy.


 


Results


An increased number of runts was observed in all TCPP-treated groups in F0 generation on PN1, which may indicate toxicity to the offspring in utero. It is noted that an increase in runts on PN1 was not observed in F1 generation or in the preliminary study, and pup weights were also not affected on PN1 in either generation. A decrease in the mean number of pups delivered was observed in the mid dose group of the F1 generation and in the high dose groups of both generations. It is not clear whether this effect is possibly due to maternal toxicity, decreased fertility of the parental animals or a developmental effect on the pups. A decrease in pup weights during the lactation period and a decrease in spleen weight were also observed in the mid and high dose groups.


It was noted that over the course of the lactation period, increasing numbers of runts were observed in the mid dose of F0 and in high dose groups of both generations. While this could be attributed to a lactational effect, it is known that pups begin to eat treated feed during the second week of the lactation period and therefore the increase in runts during the lactational period may be due to pups eating the TCPP-treated diet.  Also, as the effect on pup weight was not, or barely, observed during the first weeks of lactation, it is possibly due to consumption of TCPP-containing diets rather than a lactational effect. The numbers of pups dying in PN 1-4 could indicate enhanced toxicity of TCPP to the pups. Again, while this is possibly due to a lactational effect, the increased mortality may also be attributed to systemic toxicity to the pups in utero.  Overall, it is considered that there is no concern for a lactational effect.


 


Conclusions


Based on a weight of evidence approach, a LOAEL (developmental toxicity) of 99 mg/kg bw/day was derived, based on the increase in runts seen in F0.


This may be considered to be a relatively precautionary LOAEL, as the effect on runts was not observed in both generations.


 


Discussion


Two guideline OECD 414 studies in rat and rabbit (NTP, 2020 & van de Ven, 2018) derived NOAELs for developmental toxicity of at least 650 mg/kg bw/day (rat) and 500 mg/kg bw/day (rabbit). These were the highest doses tested, and derived in the absence of any indicators of developmental toxicity. One non-guideline pre- and post-natal study in the rat (Kawasaki et. al. 1982) derived a NOEL of ~571 mg/kg bw, based on a lack of teratogenic effects. One OECD 416 2-generation reproduction toxicity study (TNO, 2007) derived a precautionary LOAEL of 99 mg/kg bw/day based on an increased incidence of pups with decreased bodyweight (runts) in litters of the F0 generation. Further discussion of this result is offered below:


Hazards identified by EU Risk Assessment in May 2008, based on the results of the rat 2-generation study:


The EU risk assessment states: "From the same study, a LOAEL of 99 mg/kg is derived for developmental toxicity. This is based on a treatment related effect on the number of runts observed in all TCPP-treated groups of the F0 generation.


In a separate study, no treatment-related effects on foetal mortality, implantation number, resorption or foetal weight were observed following treatment of pregnant dams with TCPP. Cervical ribs and missing 13th ribs were noted at a low incidence in all treatment groups, but not in the control group. However, as a specific rib count undertaken in the 2-generation study did not reveal an increase in this effect, it is concluded that this is not toxicologically significant. Weaning rate and rearing condition were unaffected by treatment and there was no evidence of any abnormality."


 


However the registrant, in accordance with the study director does not agree with the interpretation of the 2-generation study with regard to developmental toxicity for the following reasons: The increased number of runts on PN1 is apparent in the F0 generation pups only and does not exhibit a relation with dose. The number of runts in the control group of the F0 generation pups was very low. There was no difference in the number of runts at PN1 in any of the treated groups of the F1 generation or in the DRF study. Furthermore, the group mean body weights at PN1 of the treated pups were not different from the mean weight of the control pups in the DRF study, in the F0 generation or in the F1 generation. Therefore, the higher number of runts at PN1 in the treated pups of the F0 generation should be considered an incidental finding without biological relevance and should not be interpreted as developmental toxicity.


Furthermore, no signs of developmental toxicity were observed in the dedicated though pre-GLP developmental toxicity study by Kawasaki et al 1982. Pups start eating from the diet from the second week of lactation. Therefore, the increased number of runts in the mid and dose groups towards the end of the lactation phase are either due to systemic toxicity or simply to food avoidance. Because of the appearance late in the lactation phase, this effect on the body weight of the pups is not due to lactation. For the same reason, it cannot be attributed to toxicity in utero and should therefore not be considered as developmental toxicity.


In conclusion, the changes in the incidence of runts cannot be attributed to developmental toxicity. The effect at PN1 in the F0 generation is not biologically relevant and the effect towards the end of the lactation phase should be interpreted as systemic toxicity or is due to food avoidance. In the premating phase of the F1 generation, no decrease of body weight change was observed in males or females of the low dose group. This means that the observed higher number of runts at PN21 is not associated with a persistently lower body weight in this group. For this reason, industry believes that the effect is not biologically relevant at the low dose and proposes to derive a NOAEL for systemic toxicity of 99 mg/kg bw/d.


 


Updated relevant information of March 2018:


In the 2-generation study a NOAEL of 99 mg/kg bw/day was derived for reproduction and developmental toxicity based on ‘the decreased number of pups delivered and the lower pup weight in the mid-dose group of the F1-generation’. The number of runts was not taken as basis for assessment, although statistically significantly increased in all dose groups of the F0 dams. Runts are defined in this study as pups with ‘less than mean pup weight of the control group minus 2 standard deviations’. Thus, the lower the standard deviation in the control group, the higher the number of runts may be in the treatment groups. In the pups of the F0 control dams the number of runts is 0, in the pups of the F1 control dams it is 10 (see table below). Accordingly, 14 and 11 runts in the low/high-dose F0 pups show a statistical significance whereas 17 and 14 runts in the mid/high-dose F1 pups do not. Additionally, no correlation between the number of runts and the mean pup weight on day 1 can be seen (see table below). Whereas in the F0 control pups the mean weight is 5.48g with no runts, the mean weight in the low dose group is 5.14g (14 runts) and in the mid-dose 5.31g (23 runts).


 


 





































































 Dose Group



 control



 low



 mid



 high



 


F0 runts number Day 1



 0



 


14#(7)**



 


23#(7)**



 


11#(3)



 


F0 pups mean weight day 1  



 


5.48g



 


5.14g



 


5.31g



 


5.42g



 


F0 runts number Day 21



1  



 


4(2)



 


30#(10) **



 


97#(19)#



 


F0 pups mean weight day 21



 


44.65g



 


43.23g



 


41.19g**



 


38.17g#



  


F1 runts number Day 1



 


10(4)



1



17(5) 14 (4)


 

  


F1 pups mean weight day 1



 


5.40g



 


5.58g



 


5.49g



 


5.37g



  


F1 runts number Day 21



 


5(3)



 


17** (4)



 


36#(9)



 


127#(19)#



  


F1 pups mean weight day 21



 


44.67g



 


44.57g



 


41.73g**



 


36.69g#



                    


*/**/# statistically significantly different to the control group p< 0.05/ 0.01/ 0.001


Figures in brackets represent the number of litters with pups showing the observation


 


The pup weight gain clearly decreased when the pups started eating the offered diet and with it consumed the test substance. At weaning (day 21, see table above) the number of runts was clearly increased and the body weight was significantly reduced in the mid and high-dose group. This is, however, not a classical developmental effect but a clear sign of systemic toxicity in orally exposed young animals.


In conclusion, the registrants consider derivation of a NOAEL of 99 mg/kg bw/day for developmental toxicity from this OECD 416 study to be appropriate.


 


 


Conclusions


Appraisal of the results of developmental toxicity studies with TCPP identified only one study in which potential developmental toxicity was identified (TNO, 2007). Subsequent to this study, two dedicated prenatal developmental toxicity tests in rat and rabbit (NTP, 2020 and van de Ven, 2018) were undertaken, and showed no evidence for selective developmental toxicity. Correspondingly, a non-guideline pre- and post- natal developmental toxicity study (Kawasaki et. al. 1982) reported no evidence for selective developmental toxicity. Following consideration of the evidence as part of a weight-of-evidence analysis, the registrant concludes that there is no evidence to support selective developmental toxicity following exposure to TCPP.


 


Justification for selection of Effect on developmental toxicity: via oral route:


A weight of evidence approach is followed for this endpoint. The NOAEL selected is from the OECD 414 rabbit study


 

Justification for classification or non-classification

No classification for fertility is proposed as effects that could be related to female fertility were observed in the 2-generation study only at dose levels that revealed other systemic toxicity. The effects themselves are slight (decrease in uterus weight accompanied by a slightly abnormal in estrus cycle length) and did not affect the mating indices. High exposures leading to systemic toxicity are not relevant for the exposure situation of humans and should therefore in our opinion not lead to a classification.


 


No classification is proposed for developmental toxicity and lactational effects for the following reasons: As stated above the weight of evidence from the available prenatal developmental and 2-generation reproduction study does not suggest an effect of TCPP that would be relevant for classification. Effects observed on the offspring in the 2-generation study were probably due to direct substance intake by the pups during late lactation rather than developmental effects. Following an ECHA Final Decision on a Compliance Check a pre-natal developmental toxicity study on rabbits was performed. The NOAEL for developmental effects in rabbits was determined with the highest dose tested. An NTP (2020) pre-natal developmental toxicity study (EPA OPPTS 870:3700) on rats was also undertaken. The NOAEL for developmental effects in rats was determined with the highest dose tested. 

Additional information