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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: Study generated according to OECD Test Guideline with GLP

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
One-generation toxicity study was conducted as a preliminary study
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
TCPP
There are differences in the isomer content from each supplier,but these are not important given that the properties of the isomers are expected to be very similar.
Purity
A typical purity(total of the four isomers)is>97.9%.All testing described in this report is for the commercial product.
Impurities
The impurity profile of the commercial product TCPP is specific to individual manufacturers. It is not likely that the impurities will have had particular influence on any of the results obtained.
Additives
No additives are used.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
28 Wistar rats (Crl:WI(WU)/sex/group received TCPP in the diet over two successive generations.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
not specified
Details on mating procedure:
For each mating, each female was placed with a single male from the same dose level (1:1 mating) until copulation occurs or 2 weeks have elapsed.
Each day, the females were examined for presence of sperm or vaginal plugs.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
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.
Frequency of treatment:
7-days-a-week
Details on study schedule:
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. 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 / concentrations
Remarks:
Doses / Concentrations:
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.
Basis:
nominal in diet
No. of animals per sex per dose:
28
Control animals:
yes, concurrent no treatment
yes, historical
Details on study design:
- Dose selection rationale: from preliminary study.
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked in table were included.
DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data
Estrous 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):
Parameters examined in [all] male parental generations:
[testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology]
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities]
GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: F0 and F1 males were sacrificed after at least 11 weeks of exposure.
- Maternal animals: PN21 or shortly thereafter

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1,3 and 4] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at PN21.
- These animals were subjected to postmortem examinations (macroscopic and microscopic examination) as follows: F1 and F2 pups
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
P<0.05/0.01/0.001
Reproductive indices:
Pre-coital time, mating index, and male and female fertility index between the control and TCPP treated groups were examined.
Offspring viability indices:
F0 and F1 generation live birth idex were examined.

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

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no treatment related clinical signs in parental animals in either generation.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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.
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
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.
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
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.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
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 postimplantation loss. All dams survived the delivery and there were no dams with stillborn pups in any of the groups.
ORGAN WEIGHTS (PARENTAL ANIMALS)
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.
OTHER FINDINGS (PARENTAL ANIMALS)
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
Effect level:
99 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: 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.
Dose descriptor:
NOAEL
Effect level:
85 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: Based on decreased body weights, food consumption and organ weight changes observed at mid and high dose groups.

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:
no effects observed
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)

Maternal body weights were decreased in mid and high dose animals in F1 generation during premating, in high dose F0 and F1 animals and mid dose F1 animals during gestation and in mid and high dose F1 animals during lactation. Mean food consumption was decreased in F0 and F1 females of mid and high dose groups.The mean number of pups delivered and the mean number of live pups per litter were decreased in the mid dose group of the F1 generation and in the high dose groups of both generations. These effects corrolate with a decrease in maternal body weight observed during gestation period in these dose groups and therefore could possibly be due to maternal toxicity.
Pup mortality (PN1-4) was statistically significantly increased in the low and high dose groups of F0 and in the high dose group of the F1 generation. This effect was only observed when the pup was used as the statistical unit.
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.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.
There was no effect on pup weight at PN1 in either generation.
No difference in anogenital distance of the male or female F2 pups was observed between the treated and control animals.
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).

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1 Mean terminal body weights and organ weights for males and females

 Dose (mg TCPP/kg diet) 
 Organ   Sex  0 1500 5000 15000
 Mean terminal body weight  M 388.5 370.3 381.7  359.1**
F 274.1 270.0 260.6 246.2*** 
 Mean absolute organ weight (g) 
 Liver   M   14.276   13.293   15.002   15.411 
   F   14.283   14.065   14.623   14.575 
 Brain   M   1.900   1.880   1.897   1.865 
   F   1.768   1.755   1.753   1.701 
 Adrenal   M   0.055   0.053   0.055   0.054 
   F   0.075   0.071   0.074   0.067* 
 Uterus   M   1.212   1.030*   1.092   0.982** 
 Prostate   F   0.548   0.303*   0.280**   0.286** 
 Mean organ weights relative to terminal body weight (g/kg bw) 
 Liver   M   36.733   35.891   39.291**   42.906*** 
   F   52.154   52.078   56.088   59.037* 
 Brain   M   4.898   5.090   4.975   5.207 
   F   6.459   6.503   6.736   6.924** 
 Adrenal   M   0.143   0.143   0.144   0.152 
   F   0.274   0.264   0.284   0.272 
 Uterus   F   2.004   1.121*   1.070*   1.171* 
 Prostate   M   3.131   2.795   2.858   2.735 

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

Table 2 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 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 twogeneration oral reproductive toxicity studies and 90-day studies in Wistar rats conducted at TNO between 2003 and 2007

Table 3 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 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

Table 5 Pup and Litter data from the preliminary study

 

 Dose (mg TCPP/kg diet) 

 Effect 

0

1500

 5000 

 15000 

 Total no. of pups delivered 

 98 

 96 

 92 

 98 

 Live birth index ()

 100 

 100 

 100 

 100 

 No. of pups lost (dying, missing and/ or cannibalized) on: 

 

 

 

 

 Days 1-4 

 0 

 0 

 2 

 0 

 Days 5-7 

 0 

 0 

 0 

 18** 

 Days 8-14 

 0 

 0 

 0 

 1 

 Days 15-21 

 0 

 0 

 0 

 0 

 No. pups alive Day 21 

 72 

 77 

 72 

 68** 

 Sex ratio on PN1 (M/F) 

 52/46 

 57/39 

 50/42 

 51/47 

 Mean no. of live pups per litter on PN1 

 10.89 

 9.60 

 10.22 

 9.80 

 Post implantation loss (%) 

 4.43 

 11.19 

 18.05 

 8.41 

1 All 8 pups of one dam (D71)

**Statistically significantly different to the control group (p < 0.01)

Table 6 Clinical observations in pups on Days 1-21 of lactation

 Dose (mg TCPP/kg diet) 

 0 

 1500 

 5000 

 15000 

 Runts 

 

 

 

 

 Day 1 

 0 

 4(1) 

 3(2) 

 0 

 Day 4 

 0 

 3(1) 

 1 

 20***(2) 

 Day 7 

 2(2) 

 4(1) 

 3(3) 

 26***(6) 

 Day 14 

 2(2) 

 3(2) 

 9(5) 

 50***(7) 

 Day 21 

 1 

 18***(4) 

 52***(8) 

 68***(9)*** 

 Cold pups (Day 4) 

 0 

 0 

 0 

 8**(1) 

 No milk in stomach (Day 4) 

 0 

 0 

 0 

 8**(1) 

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

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

Table 7 Delivery, pup and litter data for F0 and F1 generations

 

 Dose Group 

 Effect 

 0 

 Low 

 Mid 

 High 

 F0: 

 

 

 

 

 Mean no. of pups delivered 

 10.27 

 10.67 

 9.89 

 9.44* 

 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 ratioon 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*** 

 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

Table 8 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

Table 9 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

 

Applicant's summary and conclusion

Conclusions:
LOAEL is 99 mg/kg for female rat.
NOAEL is 85 mg/kg for male rat.
Executive summary:

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 weight 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.