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Toxicological information

Toxicity to reproduction

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Administrative data

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:
other: GLP study performed to Guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Test substance name: tris(2-chloro-1-methylethyl) phosphate
Other name: TCPP
CAS No.: 13674-84-5
Appearance: clear colourless liquid
Storage conditions: Ambient temperature

Four suppliers provided approx. 6 kg of the test substance each:
- Supresta Netherlands BV; Fyrol PCF; batch 06 130A-03-xx; expiry date 2 Jan 2008
- LANXESS Deutschland GmbH; Levagard PP; batch CH2/06016; expiry date 1 Sept 2007
- Albemarle Corp; Antiblaze TMCP; batch EJ 04AHT 051; expiry date 1 Sept 2007
- Elastogran GmbH; Lupragen TCPP; batch 00000504N0; expiry date 1 Sept 2007

The batches of the test substances were mixed in equal amounts for 30 minutes and stored in 2kg closed glass bottles. The new batch was given a TNO dispense no. 060026.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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)
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.

Target system / organ toxicity (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related

Reproductive function / performance (P1)

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.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Target system / organ toxicity (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.