p-toluenesulphonyl isocyanate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2006 October 19 - 2007 July 02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD and EU guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.35 (Two-Generation Reproduction Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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, Germany
- Age at study initiation: (P) 5-6 wks; (F1) 3 wks
- Housing: Pre-mating: Animals were housed in groups of 4 animals/sex/cage in Macrolon plastic cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages
Post-mating: Males were housed in groups of 4 animals/sex/cage in Macrolon plastic cages. Females were individually housed in Macrolon plastic cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.6-24.2
- Humidity (%): 27-95
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared for one week during the first week of study and every two weeks from Week 2 of study onwards. For the last weeks of the study, diets were prepared for a maximum of 36 days.
- Mixing appropriate amounts with Standard powder rodent diet

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling and analysis of diet preparations were performed on four occasions (week 1, 11, 22 and 33) according to the following scheme:
Group 1: accuracy (middle position of container)
Group 2: accuracy and homogeneity (top, middle and bottom position of container)
Group 3: accuracy (middle position of container)
Group 4: accuracy and homogeneity (top, middle and bottom position of container)

Duration of treatment / exposure:

F0-generation: 10 weeks prior to mating and continuing until euthanasia.
F1-generation (F0-offspring): The F1-generation was potentially exposed to the test substance in utero, through nursing during lactation and directly following weaning. After weaning, pups were treated for a minimum of 10 weeks prior to mating and continuing until euthanasia.
F2-generation (F1-offspring): The F2-generation was potentially exposed to the test substance in utero and through nursing during lactation.

Frequency of treatment:

Ad libitum

Details on study schedule:

- F1 parental animals not mated until 70 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.

Remarks:

Doses / Concentrations:
1000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
3000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
10000
Basis:
nominal in diet

No. of animals per sex per dose:

24

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on results of a 28-day toxicity study in Wistar Han rats at dose levels of 0, 1000, 5000 and 25000 ppm.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.
- Cage side observations: mortality, viability.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 of gestation, and during lactation on days 1, 4, 7, 14 and 21.

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: Yes

WATER CONSUMPTION : Yes
- Time schedule for examinations: subjective appraisal.

Oestrous cyclicity (parental animals):

Daily over a period of 3 weeks prior to pairing and throughout cohabitation

Sperm parameters (parental animals):

testis weight, epididymis weight, enumeration of homogenisation-resistant spermatids in testes and epididymis, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology.

Litter observations:

PARAMETERS EXAMINED
number and sex of pups
stillbirths
live births
presence of gross anomalies
weight gain
physical or behavioural abnormalities
The day of vaginal opening or balanopreputial separation for F1-weanlings selected for mating. As a slightly delayed balanopreputial separation and vaginal opening for high dose F1-weanlings was observed, ano-genital distance was measured on day 1 of lactation for all F2-pups.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals on day 21 post partum or shortly thereafter. Females showing no evidence of copulation were killed approximately 21 days after the last day of the mating period.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination: cervix, coagulation gland, epididymides, ovaries, prostate gland, seminal vesicles, testes, uterus, vagina, all gross lesions.
The following organs were weighed: Adrenal glands, Brain, Epididymides, Kidneys, Liver, Ovaries, Pituitary gland, Prostate, Seminal vesicles, Spleen,
Testes, Thyroid, Uterus

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at day 21 post partum or shortly thereafter.
- These animals were subjected to postmortem examinations (macroscopic and microscopic examination) as follows:
Vagina, uterus, ovaries, testis, epididymis, seminal vesicle, prostate, coagulating gland

GROSS NECROPSY
- Gross necropsy consisted of external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs with emphasis on developmental morphology.

HISTOPATHOLOGY / ORGAN WEIGTHS
The following tissues were prepared for microscopic examination: Vagina, uterus, ovaries, testis, epididymis, seminal vesicle, prostate, coagulating gland
The following tissues were weighed: brain, spleen, thymus

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 (Dunnett, 1955) (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.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 were accepted as the lowest level of significance.

Reproductive indices:

Percentage mating: Number of females mated x 100 / Number of females paired
Fertility index: Number of pregnant females x 100 / Number of females paired
Conception rate: Number of pregnant females x 100 / Number of females mated
Gestation index: Number of females bearing live pups x 100 / Number of pregnant females
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Percentage live males at First Litter Check: Number of live male pups at First Litter Check x 100 / Number of live pups at First Litter Check
Percentage live females at First Litter Check: Number of live female pups at First Litter Check x 100 / Number of live pups at First Litter Check
Percentage of postnatal loss days 0-4 post partum: Number of dead pups on day 4 post partum x 100 / Number of live pups at First Litter Check
Percentage of breeding loss day 5 until weaning: Number of dead pups between days 5 and 21 post partum x 100 / Number of live pups on day 4 post partum
Percentage live males at weaning: Number of live male pups on day 21 post partum x 100 / Number of live pups on day 21 post partum
Percentage live females at weaning: Number of live female pups on day 21 post partum x 100 / Number of live pups on day 21 post partum
Viability index: Number of live pups on day 4 post partum x 100 / Number of pups born alive
Weaning index: Number of live pups on day 21 post partum x 100 / Number of live pups on day 4 post partum

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:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
ORGAN WEIGHTS (PARENTAL ANIMALS)
Males P generation:
At 3000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (92% of control) were decreased which resulted in relative organ weight changes for the brain and kidneys (105% and 110% of control, respectively).
At 10000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (91% of control) were decreased which resulted in relative organ weight changes for the brain, liver, kidneys and seminal vesicles (108%, 108%, 118% and 116% of control, respectively).

Females P generation:
At 3000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (94% of control) were decreased which resulted in relative organ weight changes for the brain (107% of control).
At 10000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (91% of control) were decreased which resulted in relative organ weight changes for the brain, kidneys, adrenals and spleen (109%, 121%, 112% and 110% of control, respectively).

Males F1 generation:
At 3000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (93% of control) were decreased which resulted in absolute organ weight changes for the spleen (90% of control) and relative organ weight changes for the brain (108% of control).
At 10000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (87% of control) were decreased which resulted in absolute organ weight changes for the brain, liver, and spleen (95%, 89% and 89% of control, respectively), and relative organ weight changes for the brain, kidneys, seminal vesicles, testes and prostate (108%, 111%, 124%, 109% and 118% of control, respectively).

Females F1 generation:
At 3000 ppm:
Decreased body weights, body weight gain and terminal body weights at necropsy (93% of control).
At 10000 ppm:
Decreased body weights and body weight gain. Decreased food consumption during the last week of pregnancy. Terminal body weights at necropsy (85% of control) were decreased which resulted in absolute organ weight changes for the brain (93% of control) and relative organ weight changes for the brain, kidneys, adrenals, spleen and pituitary gland (110%, 118%, 112%, 113% and 120% of control, respectively).

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

1 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: Corresponds to 52-78 mg/kg bw/day for males and 75-161 mg/kg bw/day for females.

Remarks on result:

other: Generation: P and F1 (migrated information)

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

3 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: Corresponds to 165-237 mg/kg bw/day for males and 232-499 mg/kg bw/day for females.

Remarks on result:

other: Generation: F1 and F2 (migrated information)

Dose descriptor:

NOAEL

Remarks:

reproduction and breeding

Effect level:

10 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: Corresponds to 566-832 mg/kg bw/day for males and 733-1631 mg/kg bw/day for females.

Remarks on result:

other: Generation: P and F1 (migrated information)

Dose descriptor:

LOAEL

Remarks:

parental

Effect level:

3 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: Based on decreased body weights and body weight gain, and due to this organ weight changes.

Remarks on result:

other: Generation: P and F1 (migrated information)

Dose descriptor:

LOAEL

Remarks:

developmental

Effect level:

10 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: Based on decreased body weights and body weight gain, and due to this organ weight changes.

Remarks on result:

other: Generation: F1 and F2 (migrated information)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

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

BODY WEIGHT (OFFSPRING)
ORGAN WEIGHTS (OFFSPRING)
Male pups F1 generation:
At 10000 ppm:
Lower body weights for pups (terminal body weight at necropsy was 85% of control) which resulted in absolute organ weight changes for the brain, spleen and thymus (95%, 78% and 74% of control, respectively), relative organ weight changes for the brain (111% of control), and a delay in balanopreputial separation (108% of control).

Female pups F1 generation:
At 10000 ppm:
Lower body weights for pups (terminal body weight at necropsy was 84% of control) which resulted in absolute organ weight changes for the brain, spleen and thymus (94%, 78% and 73% of control, respectively), relative organ weight changes for the brain (112% of control), and a delay in vaginal opening (114% of control).

Male pups F2 generation:
At 10000 ppm:
Lower body weights for pups (terminal body weight at necropsy was 81% of control) which resulted in absolute organ weight changes for the brain, spleen and thymus (95%, 75% and 70% of control, respectively) and relative organ weight changes for the brain (117% of control).

Female pups F2 generation:
At 10000 ppm:
Lower body weights for pups (terminal body weight at necropsy was 82% of control) which resulted in absolute organ weight changes for the spleen and thymus (76% and 73% of control, respectively) and relative organ weight changes for the brain and thymus (118% and 90% of control, respectively).

Reproductive effects observed:

not specified

Table 1. Mean test article intake when corrected for mean value of recovery

Nominal dose	1000 ppm	3000 ppm	10000 ppm
Mean value of recovery	930 ppm	2820 ppm	9600 ppm
MALES F0
Pre mating	75	236	787
Post mating	52	165	568
FEMALES F0
Premating	85	264	862
Postcoitum	75	238	739
Lactation	161	499	1631
MALES F1
Premating	78	237	832
Post mating	53	165	566
FEMALES F1
Premating	86	264	887
Postcoitum	75	232	733
Lactation	160	487	1582

Table 2. Reproductive toxicity

Dose (ppm)

0

1000

3000

10000

dr

m

f

m

f

m

f

m

f

F0 animals

Mortality

no treatment-related findings

Clinical signs

no treatment-related findings

Body weight gain

dc

dc

dc

dc

mf

Food consumption

no treatment-related findings

Mating/fertility/gestation

no treatment-related findings

Oestrus cycle

no treatment-related findings

Sperm evaluation

no treatment-related findings

Organ weight

- terminal body weight

dc (94%)

dc (92%)

dc (91%)

dc (91%)

- brain

ic^r (107%)

ic^r (105%)

ic^r (109%)

ic^r (108%)

- liver

ic^r (108%)

- kidneys

ic^r (110%)

ic^r (121%)

ic^r (118%)

f

- adrenals

ic^r (112%)

- spleen

ic^r (110%)

- seminal vesicles

ic^r (116%)

Pathology

Macroscopy

no treatment-related findings

Microscopy

no treatment-related findings

F1 pups

Litter size

no treatment-related findings

Post implantation loss

no treatment-related findings

Live birth index

no treatment-related findings

Viability index

no treatment-related findings

Lactation index

no treatment-related findings

Sex ratio

no treatment-related findings

Clinical signs

no treatment-related findings

Body weight

dc

dc

Sexual maturation

ic

ic

Pup development

no treatment-related findings

Organ weight

- terminal body weight

dc (84%)

dc (85%)

- brain

dc^a (94%) ic^r (112%)

dc^a (95%) ic^r (111%)

- spleen

dc^a (78%)

dc^a (78%)

- thymus

dc^a (73%)

dc^a (74%)

Pathology

Macroscopy

no treatment-related findings

Microscopy

no treatment-related findings

F1 animals

Mortality

no treatment-related findings

Clinical signs

no treatment-related findings

Body weight gain

dc

dc

dc

dc

mf

Food consumption

dc

Mating/fertility/gestation

no treatment-related findings

Oestrus cycle

no treatment-related findings

Sperm evaluation

no treatment-related findings

Organ weight

- terminal body weight

dc (93%)

dc (93%)

dc (87%)

dc (85%)

mf

- brain

ic^r (108%)

dc^a (95%) ic^r (108%)

dc^a (93%) ic^r (110%)

- pituitary

ic^r (120%)

- liver

dc^a (89%)

- kidneys

ic^r (111%)

ic^r (118%)

- adrenals

ic^r (112%)

- spleen

dc^a (90%)

dc^a (89%)

ic^r (113%)

- testes

ic^r (109%)

- prostate

ic^r (118%)

- seminal vesicles

ic^r (124%)

Pathology

Macroscopy

no treatment-related findings

Microscopy

no treatment-related findings

F2 pups

Litter size

no treatment-related findings

Post implantation loss

no treatment-related findings

Live birth index

no treatment-related findings

Viability index

no treatment-related findings

Lactation index

no treatment-related findings

Sex ratio

no treatment-related findings

Clinical signs

no treatment-related findings

Body weight

dc

dc

Pup development

no treatment-related findings

Auditory and visual response

not performed

Organ weight

- terminal body weight

dc (81%)

dc (82%)

- brain

dc^a (95%) ic^r (117%)

ic^r (118%)

- spleen

dc^a (75%)

dc^a (76%)

- thymus

dc^a (70%)

dc^a (73%) dc^r (90%)

Pathology

no treatment-related findings

dc/ic     statistically significantly decreased/increased compared to the controls

a/r        absolute/relative organ weight

(%)       relative to control

dr         dose-related

Conclusions:

Parental toxicity was observed for both generations at the mid and high dose groups (3000 and 10000 ppm).
Developmental toxicity was observed for both generations at the high dose group (10000 ppm).
Reproduction and breeding parameters were unaffected for both generations for treatment up to 10000 ppm.
Based on these findings, the definitive parental No Observed Adverse Effect Level (NOAEL) was established as being 1000 ppm.
The definitive development NOAEL was established as being 3000 ppm.
The definitive reproduction and breeding NOAEL was established as being at least 10000 ppm.
When corrected for mean test article intake the NOAEL of 1000 ppm corresponds to 52-78 mg/kg bw/day for males and 75-161 mg/kg bw/day for females, the NOAEL of 3000 ppm corresponds to 165-237 mg/kg bw/day for males and 232-499 mg/kg bw/day for females, and the NOAEL of 10000 ppm corresponds to 566-832 mg/kg bw/day for males and 733-1631 mg/kg bw/day for females.

Executive summary:

A two-generation reproduction toxicity study of p-TSA in rats by dietary administration.

The study was based on the following guidelines:

- OECD 416, Two-Generation Reproduction Toxicity Study, January 2001.

- OPPTS 870.3800, Reproduction and Fertility Effects, August 1998.

- EC, Commission directive 2004/73/EC, B.35:"Two-generation reproduction toxicity study", April 2004.

 

After acclimatisation, male and female Wistar rats of the Fe-generation were exposed by dietary inclusion to graduated doses of the test substance. The dose levels for the F0-generation and for the F1-generation were 1000, 3000 and 10000 ppm.

At regular intervals, prepared diets were analysed for accuracy of preparation and homogeneity.

F0-males and F0-females were exposed to the test substance 10 weeks prior to mating and exposure ended at termination. F0-females were allowed to produce and rear a litter until day 21 of lactation. On day 4 of lactation litters were reduced in size to eight pups by random culling of F1-pups. After weaning, one F1-male and one F1-female of each litter were selected for cross mating with a pup of another litter of the same dose group to produce a F2-generation. Mating commenced at least 10 weeks after weaning. F1-offspring selected for mating were dosed from weaning until they were killed after weaning of the F2-offspring on day 21 of lactation. On day 4 of lactation a selection of F2-pups was culled. Pups of the F2-offspring were killed shortly after weaning.

 

All animals were subjected to daily clinical observation. Body weight and food consumption were measured over the treatment period. The regularity and duration of the estrous cycle was examined. At necropsy, macroscopic observations and organ weights were recorded. Sperm morphology, motility and count were assessed. A histopathological examination was performed on all reproduction organs and tissues. Reproduction parameters, breeding data and pup development were assessed. Blood samples were collection from F1 females (10/group) for possible measurement of thyroid hormones. These samples were discarded without analyses.

 

RESULTS

Chemical analysis revealed that the diets were prepared properly and were considered to be homogeneous.

 

The following changes were considered to be related to treatment:

F0-GENERATION

at 1000 ppm (group 2):

•      No treatment-related findings.

 

at 3000 ppm (group 3):

•      Decreased body weights and body weight gain.
Terminal body weights at necropsy were decreased which resulted in relative organ weight changes for the brain and kidneys.

 

at 10000 ppm (group 4):

•      Decreased body weights and body weight gain.
Terminal body weights at necropsy were decreased which resulted in relative organ weight changes for the brain, liver, kidneys, seminal vesicles, adrenals, and spleen.
Lower body weights for male and female pups which resulted in organ weight changes for the brain, spleen and thymus and a delay in vaginal opening and balanopreputial separation.

 

F1-GENERATION

at1000ppm (group2):

•      No treatment-related findings.

 

at3000ppm (group3):

•      Decreased body weights and body weight gain.
Terminal body weights at necropsy were decreased which resulted in organ weight changes for the brain and spleen.

 

at10000ppm (group4):

•      Decreased body weights and body weight gain.
Decreased food consumption for females during the last week of pregnancy.
Terminal body weights at necropsy were decreased which resulted in organ weight changes for the brain, liver, kidneys, seminal vesicles, adrenals, spleen, testes, prostate, and pituitary gland. Lower body weights for male and female pups which resulted in organ weight changes for the brain, spleen and thymus.

 

CONCLUSION

Treatment with p-TSA by dietary inclusion in male and female Wistar rats at dose levels of1000,3000and10000ppm revealed Fo-and F1-parental toxicity at3000and10000ppm and developmental toxicity (related to decreased body weights of dams) at10000ppm.

Reproduction and breeding parameters were unaffected for both generations for treatment up to10000ppm.

 

Based on these findings, the definitive parental No Observed Adverse Effect Level (NOAEL) was established as being1000ppm.

The definitive development NOAEL was established as being3000ppm.

The definitive reproduction and breeding NOAEL was established as being at least10000ppm.

 

When corrected for mean test article intake the NOAEL of 1000ppm corresponds to 52 -78 mg/kg bw/day for males and 75-161mg/kg bw/day for females, the NOAEL of 3000 ppm corresponds to165-237 mg/kg bw/day for males and 232 -499 mg/kg bw/day for females, and the NOAEL of 10000 ppm corresponds to 566 -832 mg/kg bw/day for males and 733 -1631 mg/kg bw/day for females.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

92 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A reliable 2-generation study according to OECD Guideline 416 was used

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A two-generation reproduction toxicity study is available with PTSA which can be read across to PTSI. This is considered justified because PTSI is extremely reactive with water and reacts instantaneously into the source substance PTSA.

 

The two-generation reproduction toxicity study with PTSA was performed in accordance with OECD416 and under GLP conditions. After acclimatisation, male and female Wistar rats of the Fe-generation were exposed by dietary inclusion to graduated doses of the test substance. The dose levels for the F0-generation and for the F1-generation were 1000, 3000 and 10000 ppm. At regular intervals, prepared diets were analysed for accuracy of preparation and homogeneity.

F0-males and F0-females were exposed to the test substance 10 weeks prior to mating and exposure ended at termination. F0-females were allowed to produce and rear a litter until day 21 of lactation. On day 4 of lactation litters were reduced in size to eight pups by random culling of F1 -pups. After weaning, one F1-male and one F1-female of each litter were selected for cross mating with a pup of another litter of the same dose group to produce a F2-generation. Mating commenced at least 10 weeks after weaning. F1-offspring selected for mating were dosed from weaning until they were killed after weaning of the F2-offspring on day 21 of lactation. On day 4 of lactation a selection of F2-pups was culled. Pups of the F2-offspring were killed shortly after weaning.

All animals were subjected to daily clinical observation. Body weight and food consumption were measured over the treatment period. The regularity and duration of the estrous cycle was examined. At necropsy, macroscopic observations and organ weights were recorded. Sperm morphology, motility and count were assessed. A histopathological examination was performed on all reproduction organs and tissues. Reproduction parameters, breeding data and pup development were assessed. Blood samples were collection from F1 females (10/group) for possible measurement of thyroid hormones. These samples were discarded without analyses.

 

Chemical analysis revealed that the diets were prepared properly and were considered to be homogeneous. In the F0-generation, no treatment-related findings were observed in the lowest dose, while at 3000 ppm decreased body weights and body weight gain were observed. In this dose group terminal body weights at necropsy were decreased which resulted in relative organ weight changes for the brain and kidneys. At the highest dose level of 10000 ppm, decreased body weights and body weight gain were observed. Terminal body weights at necropsy were decreased which resulted in relative organ weight changes for the brain, liver, kidneys, seminal vesicles, adrenals, and spleen. Furthermore, lower body weights for male and female pups were observed which resulted in organ weight changes for the brain, spleen and thymus and a delay in vaginal opening and balanopreputial separation.

In the F1-generation no treatment-related findings were observed in the lowest dose-group. At3000ppm decreased body weights and body weight gain were observed. Furthermore, terminal body weights at necropsy were decreased which resulted in organ weight changes for the brain and spleen. At 10000ppm also decreased body weights and body weight gain were observed. Furthermore, decreased food consumption for females during the last week of pregnancy was observed. Terminal body weights at necropsy were decreased which resulted in organ weight changes for the brain, liver, kidneys, seminal vesicles, adrenals, spleen, testes, prostate, and pituitary gland. Lower body weights for male and female pups were also observed which resulted in organ weight changes for the brain, spleen and thymus.

 

Based on these results it was concluded that treatment with PTSA by dietary inclusion in male and female Wistar rats at dose levels of 1000, 3000 and 10000 ppm revealed F0-and F1-parental toxicity at 3000 and 10000 ppm and developmental toxicity (related to decreased body weights of dams) at 10000 ppm. Reproduction and breeding parameters were unaffected for both generations for treatment up to10000 ppm.

The definitive parental No Observed Adverse Effect Level (NOAEL) was established as being 1000 ppm. When corrected for mean test article intake this corresponds to 52 -78 mg/kg bw/day for males and 75-161mg/kg bw/day for females (average 92 mg/kg bw/day).

The definitive development NOAEL was established as being 3000 ppm. When corrected for mean test article intake this corresponds to165-237 mg/kg bw/day for males and 232 -499 mg/kg bw/day for females (average 283 mg/kg bw/day)

The definitive reproduction and breeding NOAEL was established as being at least 10000 ppm. When corrected for mean test article intake this corresponds to 566 -832 mg/kg bw/day for males and 733 -1631 mg/kg bw/day for females (average 941 mg/kg bw/day).

The results of the 2-generation reproduction toxicity study are read across from PTSA to PTSI. The observed change leading to the NOAEL for parental toxicity at the low dose level (1000 ppm) was a decrease in body weights of < 10% at the mid-dose of 3000 ppm, which was associated with changes in relative brain, kidney and spleen weights. Although these changes have toxicological importance, they do not, by themselves indicate "significant" toxicity, as noted in Chapter 3.9.2.8.1 (a) of 1272/2008/EC. Moreover, the LOAEL of2832 mg/kg bw/day is above the guidance value for Category 2 classification after oral exposure as given in Chapter 3.9.2.9.7 of 1272/2008/EC. These reasons justify that PTSI does not need to be classified for "Specific Target Organ Toxicity (STOT) - Repeated Exposure (RE)" or "Danger of serious damage to health by prolonged exposure - R48".

Short description of key information:
NOAEL (parental), mean value calculated for males and females: 92 mg/kg bw/day
NOAEL (developmental), mean value calculated for males and females: 283 mg/kg bw/day
NOAEL (reproduction), mean value calculated for males and females: 941 mg/kg bw/day

Justification for selection of Effect on fertility via oral route:
Reliable GLP study according to OECD Guideline 416

Effects on developmental toxicity

Description of key information

NOAEL (parental): 113 mg/kg bw/day
NOAEL (developmental): 113 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See below

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See below

Qualifier:

according to guideline

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. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system 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

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

Remarks:

Doses / Concentrations:
1000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
3000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
11000 ppm
Basis:
nominal in diet

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

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.

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).

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

Dose descriptor:

NOAEL

Effect level:

3 000 ppm

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

113 mg/kg bw/day (actual dose received)

Basis for effect level:

other: developmental toxicity

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.

Abnormalities:

not specified

Developmental effects observed:

not specified

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

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).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

113 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

A reliable prenatal developmental toxicity study according to OECD Guideline 414 was used

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

A developmental toxicity study with rabbits is available for PTSA, which can be read across to PTSI. This is considered justified because PTSI is extremely reactive with water and reacts instantaneously into the source substance PTSA.

In a developmental toxicity study which was performed in accordance with OECD 414 and under GLP-conditions, 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.

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.

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-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).

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 PTSA 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 PTSA 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 PTSA or to a combination of PTSA 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 PTSA was established as being 3000 ppm (113 mg/kg body weight/day).

Justification for selection of Effect on developmental toxicity: via oral route:
A reliable prenatal developmental toxicity study according to OECD Guideline 414 was used

Justification for classification or non-classification

Based on the available information from the 2-generation reproduction toxicity study and the prenatal developmental toxicity study, PTSI does not have to be classified as reprotoxic/toxic to the development in accordance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2008/EEC.