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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007, March 14 - July 27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD and EU guidelines and according to GLP principles. In principle an evaluation of two separately treated cohorts.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See below
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See below
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
Protocol deviations:
1 On 05 February 2007 (Dose range finding study, NOTOX Project 474086, see Appendix 5) and on 24 March 2007 (Main study), no water consumption was determined. Evaluation: Sufficient data available for evaluation of effects on water consumption.
2 No skeletal examinations were performed on fetuses 03 and 04 from animal 31. Thorough examination was not possible as skeletal preparations were fallen into individual bones, possibly due to the use of an incorrect fixative. Evaluation: The examination that was performed did not reveal findings related to treatment. There were sufficient fetuses left in the litter for evaluation.
3 On 17 and 23 July 2007 animals have been observed, however observations have not been entered into the computer. On both days there were no remarkable changes when compared to the observation on the day before. Evaluation: Animals were observed, only no on-line data is available.
4 Temporary deviations from the minimum and maximum level of temperature occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations.
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): p-TSA
- Substance type: White crystalline powder
- Physical state: Solid
- Analytical purity: 99.9%
- Impurities (identity and concentrations): 0.1% water
- Purity test date: 2006 July 28
- Lot/batch No.: 0603514009
- Expiration date of the lot/batch: 2008 March 31
- Stability under test conditions: p-TSA diets at target concentrations of 1000 and 11000 ppm were determined to be stable during storage for at least 53 days at room temperature without protection from light.
- Storage condition of test material: At room temperature in the dark in tightly closed containers. Keep dry.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan France SARL, Gannat, France
- Age at study initiation: Part I: Females were approximately 18-20 weeks. Part II: Females were approximately 21-23 weeks.
- Weight at study initiation: Day 0 post coitum: 2478-3887 g
- Fasting period before study: Not applicable
- Housing: Females were individually housed in labelled cages with perforated floors
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Part I: At least 13 days prior to insemination. Part II: At least 3 weeks prior to insemination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.9-24.1°C. Temporary deviations from the minimum and maximum level of temperature occurred. Laboratory historical data do not indicate an effect of the deviations.
- Humidity (%): 31 - 81%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Milli-U water (approximately 12% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage.

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared at least once every two weeks.
- Mixing appropriate amounts with Standard powder rabbit diet
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were analyzed using 30 and 60 minutes ultrasonication in Part I and using 60 minutes ultrasonication in Part II. The concentrations analysed in diets of Group 2, Group 3 and Group 4 were between 80 and 108% (Part I) using 30 minutes of ultrasonication and between 86% and 114% (Part I) and 81% to 106% (Part II) of target using 60 minutes of ultrasonication for extraction. For studies using diet, a range of 80-120% is considered acceptable.
Diets are considered to be homogeneous with a coefficient variation <10%.
Details on mating procedure:
Not applicable. Animals are inseminated.
Duration of treatment / exposure:
Day 7 - 29 post coitum.
Frequency of treatment:
Ad libitum
Duration of test:
30 days
No. of animals per sex per dose:
27 at 0 and 3000 ppm (24 in part I, 3 in part II)
30 at 1000 and 11000 ppm (24 in part I, 6 in part II)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on a range finding study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes : mortality
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 0 post-coitum onwards.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 4, 7, 10, 13, 16, 20, 23, 26 and 29 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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: Yes
Days 0-4, 4-7, 7-10, 10-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily from Day 0 post-coitum onwards.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 29 post-coitum
- Organs examined: External, thoracic and abdominal examinations, ovary and uterine horn, female genital tract including the placentas
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- The number of corpora lutea (ovaries in situ)
- The weight of the gravid uterus
- The number and distribution of live and dead foetuses
- The number and distribution of embryo-foetal deaths
- The weight of each foetus
- The sex of each foetus (during further foetal examination)
- Externally visible macroscopic foetal abnormalities.
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [all per litter ]
- Skeletal examinations: Yes: [all per litter ]
- Head examinations: Yes: [one-half of the foetuses per litter ]
Statistics:
The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex (Dunett, 19955).
• The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data could not assumed to follow a normal distribution (Miller, 1981).
• The Fisher-exact test was applied to frequency data (Fisher, 1950).
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Indices:
Pre-implantation loss: (Number of corpora lutea - number of implantation sites) x 100 /Number of corpora lutea
Post-implantation loss: (Number of implantation sites - number of live foetuses) x 100 /Number of implantation sites


Historical control data:
Historical control data on the background incidence of foetal malformations and developmental variations in New Zealand White rabbits from the same source was used for comparison with concurrent controls in the study.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At the high dose group (11000 ppm), decreased body weight and body weight gain were noted, which were most pronounced on the first days of treatment. Although an increase in body weight and body weight gain was noted from Day 10 post-coitum onwards, body weight remained lower for the entire treatment period. Body weight gain at 11000 ppm was also reduced at necropsy after correction for uterus weight. In addition, reduced food consumption was noted mainly during the first days of treatment (Days 7-10 post-coitum). Food consumption improved from Day 10 post-coitum onwards and had returned to control values at the end of treatment. The findings in body weight and food consumption were accompanied by a reduction in water consumption from Day 7 post-coitum onwards, which increased to normal values at the end of treatment.

At the intermediate dose group (3000 ppm), similar effects on body weight, food- and water consumption were noted during the first days of treatment. However, these effects were less severe and recovered sooner and were therefore considered to be transient.

No maternal toxicity was observed at the low dose group (1000 ppm).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
113 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No effects on pre- and post implantation loss, litter size and sex ratio were noted.
A low pregnancy rate was observed during Part I of the study. As treatment started after implantation and as the low pregnancy rate was observed in all groups, including the control group, in absence of a dose response relationship, the low pregnancy was not treatment related. The low pregnancy rate might be due to stress caused by periods of loud noises due to on-site construction work.
In both Parts I and II and after combining data of these parts, a trend towards reduced body weight of fetuses (statistically not significant) was noted at the high dose group (11000 ppm). This was considered to be related to the decreased maternal body weight at 11000 ppm.
The total number of malformations observed in fetuses (litters) in the different treatment groups were 3(3), 0(0), 4(3) and 11(8) in the control, 1000, 3000 and 11000 ppm groups, respectively. The only treatment related malformations were vertebral anomalies with or without associated rib anomaly, which were noted in 4 fetuses from four different litters in Part I. After combining data of both Part I and II, a statistically significant increase in the mean litter proportion of fetuses with vertebral anomaly with or without associated rib anomaly, a skeletal malformation, was noted at a dose level of 11000 ppm, whilst this finding was not observed for fetuses in the control, 1000 and 3000 ppm groups. In addition, no such findings were observed in Part II of the study.
Vertebral anomalies with or without associated rib anomaly had not been seen in the concurrent control group. Historical control data revealed that these anomalies had occurred at a fetal incidence of 0.4% in control animals (range 0.0% – 1.2%).
To further determine the toxicological significance of vertebral anomalies with or without associated rib anomaly, all fetuses of the dose range finding study were skeletally examined. Results of skeletal evaluations did not show vertebral anomalies with or without associated rib anomaly up to a dose level of 20000 ppm.
Based on the findings in this current study, a direct fetal effect of the test substance at 11000ppm in the diet to cause vertebral anomalies with or without associated rib anomaly can not be ruled out. However it might also be related to increased maternal stress in these test substance treated groups, since it was not observed in any of the Part II animals in this study, or in animals dosed with up to 20000 ppm in the dose ranging study.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: - Statistically significant increase in the mean litter proportion of vertebral anomalies with or without associated rib anomaly at 11000 ppm.
Dose descriptor:
NOAEL
Effect level:
113 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

The table below shows the incidence of vertebral anomalies with or without associated rib anomaly in the main and dose range findings study, when compared to the total number of fetuses and litters (as well as total malformations and developmental delay):

Part I

Fetuses (Litter) with Finding

Total # Fetuses

Total # Litters

A

B

C

Control

1(1)

0(0)

0(0)

93

17

1000 ppm

0(0)

0(0)

0(0)

96

15

3000 ppm

3(2)

0(0)

2(1)

104

17

11000 ppm

11(8)

4(4)

3(3)

88

14

Part II and Ranger Combined

Fetuses (Litter) with Finding

Total # Fetuses

Total # Litters

A

B

C

Control

2(2)

0(0)

0(0)

52

8

1000 ppm

0(0)

0(0)

1(1)

14

3

3000 ppm

1(1)

0(0)

0(0)

17

3

5000 ppm

2(2)

0(0)

0(0)

44

6

10000/11000 ppm

1(1)

0(0)

0(0)

66

12

20000 ppm

0(0)

0(0)

0(0)

32

4

 

A = Total malformations

B = Malformations: vertebral anomalies with or without associated rib anomaly

C = Developmental delay: Vertebral centra anomalies

Applicant's summary and conclusion

Conclusions:
Rabbits dosed with 11000 ppm p-TSA showed evidence of toxicity with reduced body weight which remained lower than controls for the entire treatment period. Body weight gain at 11000 ppm was also reduced at necropsy after correction for uterus weight. Transient effects on body weight were noted at 3000 ppm. No treatment related toxicity was observed at 1000 ppm.

Based on the results obtained in this prenatal developmental toxicity study, it is concluded that dietary administration of p-TSA to pregnant rabbits during the period of organogenesis at a high dose level of 11000 ppm was associated with a statistically significant increase in the mean litter proportion of vertebral anomalies with or without associated rib anomaly. However, whether this was related to p-TSA or to a combination of p-TSA with incidental stress which occurred during the first part of the study could not be determined, since the findings were not observed in any of the Part II animals in this study, or in animals dosed up to 20000 ppm in the dose ranging study. No test substance related findings on fetal morphology occurred at dose levels of 1000 and 3000 ppm. Therefore, a dosage level of 3000 ppm (113 mg/kg body weight/day) was considered to be the No Observed Adverse Effect Level (NOAEL) for embryo/fetal developmental toxicity.

Furthermore, based on the effects on body weight, food and water consumption, the maternal No Observed Adverse Effect Level (NOAEL) for p-TSA was established as being 3000 ppm (113 mg/kg body weight/day).
Executive summary:

STUDY OUTLINE

Four groups of 27 to 30 New Zealand White rabbits were inseminated (Day 0 post-coitum) and exposed by dietary exposure to 0, 1000, 3000 and 11000 ppm (equivalent to 0, 41, 113 and 367 mg/kg body weight/day respectively) from Days 7 to 29 post-coitum.

The study consists of Part I and II. In Part I of the study 24 animals/group were inseminated. A low pregnancy rate was observed in all groups, which resulted in an insufficient number of litters; seventeen litters in the control and mid dose group, fifteen litters in the low dose group and fourteen litters in the high dose group. Therefore, 18 additional animals (3 animals/group in Groups 1 and 3, and 6 animals/group in Groups 2 and 4) were added to the study (Part II), which resulted in the addition of three litters in the control, low and mid dose group and six litters in the high dose group. In Part I and II, treatment and study procedures were comparable.

EVALUATED PARAMETERS

Females were checked daily for the presence of clinical signs. Body weight and food consumption of females was determined at periodic intervals; and water consumption daily. Diet analysis was performed on prepared diets.

All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The ovaries and uterine horns were dissected and examined for the number of corpora lutea, the weight of the gravid uterus, the number and distribution of live/dead fetuses and embryo-fetal deaths, the weight of each fetus, fetal sex and externally visible fetal macroscopic abnormalities. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’ fixative and subsequently sliced, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous alcohol and stained with Alizarin Red S for skeletal examinations.

RESULTS

Accuracy, homogeneity and stability of diet preparations were demonstrated by analyses.

Maternal findings

At the high dose group (11000 ppm), decreased body weight and body weight gain were noted, which were most pronounced on the first days of treatment. Although an increase in body weight and body weight gain was noted from Day 10 post-coitum onwards, body weight remained lower for the entire treatment period. Body weight gain at 11000 ppm was also reduced at necropsy after correction for uterus weight. In addition, reduced food consumption was noted mainly during the first days of treatment (Days 7-10 post-coitum). Food consumption improved from Day 10 post-coitumonwards and had returned to control values at the end of treatment. The findings in body weight and food consumption were accompanied by a reduction in water consumption from Day 7 post-coitum onwards, which increased to normal values at the end of treatment.

At the intermediate dose group (3000 ppm), similar effects on body weight, food- and water consumption were noted during the first days of treatment. However, these effects were less severe and recovered sooner and were therefore considered to be transient.

No maternal toxicity was observed at the low dose group (1000 ppm).


Developmental findings

No effects were noted on pre- and post implantation loss, litter size and sex ratio.

A low pregnancy rate was observed during Part I of the study. As treatment started after implantation and as the low pregnancy rate was observed in all groups including the control group in absence of a dose response relationship, the low pregnancy was not treatment related. The low pregnancy rate might be due to stress caused by periods of loud noises due to construction work.

In both Parts I and II and after combining data of these parts, a trend towards reduced body weight of fetuses (statistically not significant) was noted at the high dose group (11000 ppm). This was considered to be related to the decreased maternal body weight at 11000 ppm.

The total number of malformations observed in fetuses (litters) in the different treatment groups were 3(3), 0(0), 4(3) and 11(8) in the control, 1000, 3000 and 11000 ppm groups, respectively. The only treatment related malformations were vertebral anomalies with or without associated rib anomaly, which were noted in 4 fetuses from four different litters in Part I. After combining data of both Part I and II, a statistically significant increase in the mean litter proportion of fetuses with vertebral anomaly with or without associated rib anomaly, a skeletal malformation, was noted at a dose level of 11000 ppm, whilst this finding was not observed for fetuses in the control, 1000 and 3000 ppm groups. In addition, no such findings were observed in Part II of the study.

Vertebral anomalies with or without associated rib anomaly had not been seen in the concurrent control group. Historical control data revealed that these anomalies had occurred at a fetal incidence of 0.4% in control animals (range 0.0% – 1.2%).

To further determine the toxicological significance of vertebral anomalies with or without associated rib anomaly, all fetuses of the dose range finding study were skeletally examined (NOTOX Project 474086, Appendix 7C). Results of skeletal evaluations did not show vertebral anomalies with or without associated rib anomaly up to a dose level of 20000 ppm.

The table below shows the incidence of vertebral anomalies with or without associated rib anomaly in the main and dose range findings study, when compared to the total number of fetuses and litters:

Part I:

finding

fetuses (litters)

Total number of fetuses

Total number of litters

Control:

0 (0)

93

17

1000 ppm

0 (0)

96

15

3000 ppm

0 (0)

104

17

11000 ppm

4 (4)

88

14

Part II and range finding combined:

finding

fetuses (litters)

Total number of fetuses

Total number of litters

Control:

0 (0)

52

8

1000 ppm

0 (0)

14

3

3000 ppm

0 (0)

17

3

5000 ppm

0 (0)

44

6

10000/11000 ppm

0 (0)

66

12

20000 ppm

0 (0)

32

4

Based on the findings in this current study, a direct fetal effect of the test substance at 11000 ppm in the diet to cause vertebral anomalies with or without associated rib anomaly can not be ruled out. However it might also be related to increased maternal stress in these test substance treated groups, since it was not observed in any of the Part II animals in this study, or in animals dosed with up to 20000 ppm in the dose ranging study.

CONCLUSION

Rabbits dosed with 11000 ppm p-TSA showed evidence of toxicity with reduced body weight which remained lower than controls for the entire treatment period. Body weight gain at 11000 ppm was also reduced at necropsy after correction for uterus weight. Transient effects on body weight were noted at 3000 ppm. No treatment related toxicity was observed at 1000 ppm.

Based on the results obtained in this prenatal developmental toxicity study, it is concluded that dietary administration of p-TSA to pregnant rabbits during the period of organogenesis at a high dose level of 11000 ppm was associated with a statistically significant increase in the mean litter proportion of vertebral anomalies with or without associated rib anomaly. However, whether this was related to p-TSA or to a combination of p-TSA with incidental stress which occurred during the first part of the study could not be determined, since the findings were not observed in any of the Part II animals in this study, or in animals dosed up to 20000 ppm in the dose ranging study. No test substance related findings on fetal morphology occurred at dose levels of 1000 and 3000 ppm. Therefore, a dosage level of 3000 ppm (113 mg/kg body weight/day) was considered to be the No Observed Adverse Effect Level (NOAEL) for embryo/fetal developmental toxicity.

Furthermore, based on the effects on body weight, food and water consumption, the maternal No Observed Adverse Effect Level (NOAEL) for p-TSA was established as being 3000 ppm (113 mg/kg body weight/day).