Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
No other data available.
Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
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:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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).

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
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).


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).

Dose descriptor:
NOAEL
Remarks:
parental
Generation:
F1
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
Generation:
F1
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
Generation:
F1
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
Generation:
F1
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.
Dose descriptor:
LOAEL
Remarks:
developmental
Generation:
F1
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.
BODY WEIGHT (OFFSPRING)
ORGAN WEIGHTS (OFFSPRING)

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

icr

(107%)

icr

(105%)

icr

(109%)

icr

(108%)

- liver

icr

(108%)

- kidneys

icr

(110%)

icr

(121%)

icr

(118%)

f

- adrenals

icr

(112%)

- spleen

icr

(110%)

- seminal vesicles

icr

(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

dca (94%)

icr

(112%)

dca (95%)

icr

(111%)

- spleen

dca (78%)

dca (78%)

- thymus

dca (73%)

dca (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

icr (108%)

dca (95%)

icr (108%)

dca (93%)

icr (110%)

- pituitary

icr (120%)

- liver

dca (89%)

- kidneys

icr (111%)

icr (118%)

- adrenals

icr (112%)

- spleen

dca (90%)

dca (89%)

icr (113%)

- testes

icr (109%)

- prostate

icr (118%)

- seminal vesicles

icr (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

dca (95%)

icr

(117%)

icr

(118%)

- spleen

dca (75%)

dca (76%)

- thymus

dca (70%)

dca (73%)

dcr

(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, 3000 and 10000 ppm revealed Fo-and F1-parental toxicity at3000and10000ppm and developmental toxicity (related to decreased body weights of dams) at 10000ppm.

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 75-161mg/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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
165 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
There are two studies available (OECD 422 screening study, and a two-generation study), both of high quality. The two-generation study is selected as the most relevant for the evaluation of reproduction toxicity. The selected NOAEL represents the lowest reproduction/development NOAEL of 3000 ppm (corresponding to 165-237 mg/kg bw/day in males and 232 -499 mg/kg bw/day in females)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There are two studies available for evaluation of reproduction toxicity:

70-55-3, Toxicity to reproduction 2-gen rat, NOTOX 474064, 2007

A full two-generation study in rats has been performed under GLP and according to OECD 416 with p-TSA administration by dietary admixture at levels of 0, 1000, 3000 and 10,000 ppm.

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.

No effects were observed at 1000 ppm. At 3000 ppmterminal body weights of F0 and F1 parent animals at necropsy were decreased which resulted in relative organ weight changes for the brain and kidneys.

At 10,000 ppm the terminal body weights at necropsy were decreased which resulted in relative organ weight changes for the brain, liver, kidneys, seminal vesicles, adrenals, and spleen. In the F1 generation also testes, prostate, and pituitary gland showed relative organ weight changes. Also 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 balano-preputial separation in F1 animals.

Based on these findings thestudy concluded upon

- NOAEL parental of1000ppm (males52 -78 mg/kg bw/day; females75-161 mg/kg bw/day).

- NOAEL developmental toxicity of3000ppm(males 165-237 mg/kg bw/day; females232 -499 mg/kg bw/day)

- NOAEL reproduction and breeding of at least 10000ppm (males 566 -832 mg/kg bw/day; females 733 -1631 mg/kg bw/day)

70-55-3, Toxicity to reproduction, MHW Japan, 1994

OECD 422, Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test in rat by gavage under GLP. Dose levels: 0 (vehicle, 5% Arabic gum), 120, 300, 750 mg/kg/day, with 13 animals per dose group per sex. Dosing period: Females, from 14 days before mating to day 3 of lactation. Males 42 days.

Mating performance and fertility were not affected by the test compound. Two of 10 females in the high-dose group showed signs of difficult delivery and all of their offspring died by day 3 of lactation.

Reproduction parameters (i.e., duration of gestation, numbers of corpora lutea, implantations and resorptions, litter size, and sex distribution) were comparable among all four groups, including the control. However, significant decreases in both survival rate and body weight of newborns on day 0 of lactation were observed in the high-dose group. Morphological observations of offspring revealed no teratogenic effects of 4-methylbenzenesulfonamide.

NOEL for P generation: 300 mg/kg/day

NOEL for F1 generation: 300 mg/kg/day

Both available studies do not indicate a specific concern regarding reproduction toxicity. Reproductive effects that have been observed at highest dose levels can bet attributed to the lower parental condition as indicated by the lower body weight, and subsequent lower body weight of the pups. This in turn can affect development in pups as indicated by observeddelay in vaginal opening and balano-preputial separation. No effects were seen on fertility and breeding results.

The LOAEL from the two-generation study is at 10,000 ppm (males566 -832 mg/kg bw/day; females733 -1631 mg/kg bw/day), which compares well with the reproduction LOAEL of 750 mg/kg bw/day by gavage from the OECD 422 study.

The overall NOAEL for reproduction toxicity is set at 3000 ppm (males 165-237 mg/kg bw/day; females232 -499 mg/kg bw/day), a level that correlates to the level at which parental toxicity becomes visible. This level is also at the same order of magnitude as the NOAEL of 300 mg/kg bw/day observed in the OECD 422 study following dosing by gavage.


Short description of key information:
Available studies do not indicate a specific concern regarding reproduction toxicity. Reproductive effects that have been observed at highest dose levels can be attributed to deteriorating parental condition as indicated by the lower body weight, and subsequent lower body weight of the pups. The selected NOAEL represents the lowest reproduction/development NOAEL of 3000 ppm from the available 2-generation study (corresponding to 165-237 mg/kg bw/day in males and 232 -499 mg/kg bw/day in females).

Justification for selection of Effect on fertility via oral route:
Only one two-generation study available of high quality.

Justification for selection of Effect on fertility via inhalation route:
Testing by the inhalation route is not appropriate since exposure of humans via inhalation is not likely taking into account the vapour pressure of the substance and/or the possibility of exposure to particles or droplets of an inhalable size. The vapour pressure is very low (<0.000286 Pa at 20°C), the crystalline solid is of low dustiness (the respirable fraction (≤ 4 µm) is 0%), and the pattern of use does not lead to the formation of droplets of an inhalable size. In addition the substance is of general low toxicity and not irritating. Testing by inhalation is therefore not necessary.

Effects on developmental toxicity

Description of key information

There is one recent GLP full OECD 414 study available in rats showing no teratogenic effects at toxic dose levels pf TSA (o'p-Toluene sulphonamide)

There is one recent GLP full OECD 414 comparable study available in rats showing no teratogenic effects at toxic dose levels of TSA (o,p -Toluene sulphonamide). Results are well comparable to the results on p-TSA from an OECD 414 study with rabbits (see below). A NOAEL of 50 mg/kg bw/d was westablished for maternal and developmental toxicity.

A NOAEL of 50 mg/kg bw/d was established for maternal and developmental toxicity.

In an OECD 414 study in rabbits there was an increase observed in the mean litter proportion of vertebral anomalies with or without associated rib anomaly at maternal toxic dose level of 11,000 ppm (equivalent to 367 mg/kg body weight/day). However, the relevance of this finding is questionable as in part II of the study these effects were not observed, even at much higher dose levels. No test substance related findings on foetal morphology occurred at dose levels of 1000 and 3000 ppm. Consequently the NOAEL for both maternal and foetal toxicity is set at 3000 ppm (114 mg/kg bw/day). In an additional OECD 422 reproduction screening study evaluation of offspring for visceral and skeletal malformation was performed. This study indicated no teratogenic effects. Also the OECD 416 two-generation study resulted to a NOAEL for development of 3000 ppm (corresponding to 165-237 mg/kg bw/day for males and 232-499 mg/kg bw/day for females) based on decreased body weights of pups at 10000 ppm. There is one recent GLP full OECD 414 comparable study available in rats showing no teratogenic effects at toxic dose levels of TSA (o,p -Toluene sulphonamide).

A NOAEL of 50 mg/kg bw/d was established for maternal and developmental toxicity.

The results with o/p-TSA did not show a teratogenic effect when administered orally to mated rats at 500 mg/kg/day and less. A NOAEL of 50 mg/kg bw/d was established for maternal and developmental toxicity.

The results with o/p-TSA did not show a teratogenic effect when administered orally to mated rats at 500 mg/kg/day and less. A NOAEL of 50 mg/kg bw/d was established for maternal and developmental toxicity.

The results with o/p-TSA did not show a teratogenic effect when administered orally to mated rats at 500 mg/kg/day and less. A NOAEL of 50 mg/kg bw/d was established for maternal and developmental toxicity.

Based on these results, o/p-TSA did not produce a teratogenic effect when administered orally to mated rats at 500 mg/kg/day and less. A NOAEL of 50 mg/kg bw/d was established for maternal and developmental toxicity.

Based on these results, o/p-TSA did not produce a teratogenic effect when administered orally to mated rats at 500 mg/kg/day and less. A NOAEL of 50 mg/kg bw/d was established for maternal and developmental toxicity.

There is one recent GLP full OECD 414 comparable study available in rats showing no teratogenic effects at toxic dose levels of TSA (o,p -Toluene sulphonamide)

In an OECD 414 study in rabbits there was an increase observed in the mean litter proportion of vertebral anomalies with or without associated rib anomaly at maternal toxic dose level of 11,000 ppm (equivalent to 367 mg/kg body weight/day). However, the relevance of this finding is questionable as in part II of the study these effects were not observed, even at much higher dose levels. No test substance related findings on foetal morphology occurred at dose levels of 1000 and 3000 ppm. Consequently the NOAEL for both maternal and foetal toxicity is set at 3000 ppm (114 mg/kg bw/day).
In an additional OECD 422 reproduction screening study evaluation of offspring for visceral and skeletal malformation was performed. This study indicated no teratogenic effects. Also the OECD 416 two-generation study resulted to a NOAEL for development of 3000 ppm (corresponding to 165-237 mg/kg bw/day for males and 232-499 mg/kg bw/day for females) based on decreased body weights of pups at 10000 ppm.

Link to relevant study records

Referenceopen allclose all

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.
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
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
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
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
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.
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
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 (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

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).

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
18 July 1984 - 15 August 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed under GLP and according to a method similar to currently internationally accepted guidelines. No verification of dose levels, and no information on food consumption. Exposure from implantation (day 6) through day 15 of gestation instead of from implantation until one day prior to the day of scheduled kill (day 19). Gravid uterus weight was not determined.
Justification for type of information:
See attached document: Support Cross-reading - Toluenesulphonamides 20130329
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Performed in accordance with Series 83-3 of the Environmental Protection Agency Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, issued November, 1982.
Devations:
- Gravid uterus weight was not determined.
- No verification of dose levels
- No information on food consumption.
- Exposure from implantation (day 6) through day 15 of gestation instead of from implantation until one day prior to the day of scheduled kill (day 19).
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: COBS CD
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation: 70 days
- Weight at study initiation: The rats were 118 days old at the time of mating and weighed between 255 and 346g on gestation day 0.
- Fasting period before study: Not applicable
- Housing: The rats were housed individually in suspended wire-mesh cages from receipt until sacrifice except during mating when one male and one female were placed together.
- Diet (e.g. ad libitum): ad libitum, basal Laboratory diet of Purina Certified Rodent Chow #5002
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 48 day acclimation period


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ± 0.35
- Humidity (%): 67 ± 13.4
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: 18 July 1984 - 15 August 1985
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of Santicizer 9 for each group was ground daily with a mortar and pestle, weighed, and suspended i n the vehicle, Mazola corn oil, using a tissue homogenizer. The test material suspension was then transferred to a graduated mixing cylinder and additional vehicle added to yield a sufficient volume of prepared test material.
The suspension was shaken by hand and dispensed into a capped container. Prior to administration, the suspension was stirred using a magnetic stit
bar and stir plate.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: The test article was prepared at concentrations to permit the administration of dosage levels of 50, 250 and 500 mg/kg/dap at a dosage volume of 10 ml/kg. Individual dosages were determined from individual body weights recorded on gestation days 6, 9 and 12.
- Amount of vehicle (if gavage): max. 10 ml/kg.
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- After "no data" days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none
Duration of treatment / exposure:
single daily dose on days 6 through 15 of gestation
Frequency of treatment:
Single daily dose
Duration of test:
Days 6 through 15 of gestation
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: range finding study. For summary see "1333-07-9, Developmental toxicity / teratogenicity, Leng, 1985, RS"
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Prior to treatment, the females were observed twice daily for mortality and overt changes in appearance and behavior. They were
observed twice daily for mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily on days 6 through 15 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0, 6, 9, 12, 16 and 20.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: The abdominal and thoracic cavities and organs of the dams wesere examined for grossly evident morphological changes and the carcasses discarded. Maternal tissues vere preserved in 10% neutral buffered formalin for possible histopathological examination as deemed necessary by the gross findings.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The number and location of viable and nonviable fetuses, uteri from females that appeared nongravid uere opened and placed in 10% anmonium sulfide solution for detection of implantations.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litte
- Head examinations: No: included in soft tissue examination, only applicable for non-rodents
Statistics:
All statistical analyses compared the treatment groups with the control group, with the level of significance at P<0.05 and p<0.01. All means were accompanied by standard deviations. Male to female fetal sex ratios and the proportions of litters with malformations were compared using the Chi-square test criterion with Yates' correction for 2 x 2 contingency tables and/or Fisher's exact probability test as described by siegel to judge significance of differences.
The proportions of resorbed and dead fetuses and postimplantation losses were compared by the Mann-Whitney U-test as described by Siegel and Weil to judge significance of differences.
Mean numbers of corpora lutea, total implantations, live fetuses, mean fetal body weights, and mean maternal body weights and body weight changes were compared by analysis of variance (one-way classification). Barelett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie using Dunnet t 's multiple comparison tables to judge significance of differences.
Historical control data:
Included in the study
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Survival was 100% among the control, 50, 250 and 500 mg/kg/day groups. The test article did not produce meaningful differences in maternal appearance and behavior at any dosage level. There was no test item related effect on pregnancy rate.
Clinical signs observed in some groups inclusive of the control, included the following : alopecia .and scabbing, sparse haircoat,
and malligned upper incisors with associated ocular discharge.
The following gross changes were noted during necropsy in the treated and control groups: hydronephrosis with or without distended ureters, misshapen kidney, distended uterus and cervix, and adherence of the liver to the kidney.
The test article had a marked inhibitory effect on maternal body weight gain at 250 and 500 mg/kg/day during the overall treatment interval.Moreover, marked reductions in gain were evident at 500 mg/kg/day during the overall gestation interval. Weight loss was observed during the gestation day 6 to 9 subinterval for both groups. All of these decreases were statistically significant. No similar effect was observed at 50 mg/kg/day.
An increase in postimplantation loss was noted for the 250 and 500 mg/kg/day groups, relative to the control. The increase was statistically significant at 500 mg/kg/day. The test article had no effect on the i.e., corpora lutea, total implantations, viable litter size, and sex Distribution, at 500 mg/kg/day and less.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
50 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:
A treatment-related trend of fetal body weight inhibition was observed at all dosage levels. Fetal body weights were statistically significantly less than the control at 250 and 500 mg/kg/day. Weight inhibition, although very slight and not statistically significant, was also observed at 50 mg/kg/day and was deemed compound related as it was considered part of the trend observed at higher dosage levels.
The incidence of fetal malformations occurring in the treated groups was not meaningfully different from the control. A single instance each of a tail anomaly with anal atresia, and a vertebral anomaly occurred in a 250 mg/kg/day litter. Also, one 500 mg/kg/day fetus exhibited a retroesophageal aortic arch. No malformations occurred in the control and 50 mg/kg/day animals.
With respect to developmental variations, the number of litters exhibiting unossification of sternebrae #5 and/or #6 was increased exclusively at 500 mg/kg/day relative to the control. The remaining variations were noted in the control and treated groups at similar incidence or occurred as an isolated incidence and were deemed biologically irrelevant.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: Significant effect in th fetuses were only observed at dose level where maternal toxicty was also observed.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Based on these results, o/p-TSA did not produce a teratogenic effect when administered orally to mated Charles River COBS CD rats at 500 mg/kg/day and less.
Executive summary:

A study was performed under GLP and according to a method similar to currently internationally accepted guidelines. Performed in accordance with Series 83-3 of the Environmental Protection Agency Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, issued November, 1982. Mated Charles River COBS CD rats consecutively assigned to one control and three treatment groups of 25 animals each were used to determine the teratogenic potential of Santicizer 9. Dosage levels of 50, 250 and 500 mg/kg/day were administered orally by gavage as a single daily dose on days 6 through 15 of gestation at a constant volume of 10 ml/kg. The control group received the vehicle only, corn oil, on a comparable regimen. Cesarean sections were performed on all surviving females on gestation day 20 and the fetuses were removed for teratologic evaluation.

Survival was 100% among all groups on study. Santicizer 9 produced meaningful changes in maternal body weight gain and several Cesarean section parameters when administered at 250 and 500 mg/kg/day. Maternal weight loss was evident during a portion of the treatment interval at 250 and 500 mg/kg/day and both groups exhibited significant (p<0.01) reductions in weight gain during the overall treatment period. Statistically significant reductions were also noted at 500 mg/kg/day during the overall gestation period. Fetotoxicity was apparent at 250 and 500 mg/kg/day as evidenced by increased postimplantation loss which was statistically significant at 500 mg/kg/day. In addition, a treatment-related trend of fetal body weight inhibition was noted at all dosage levels and values were significantly different from the control, p<0.01 at 250 and 500 mg/kg/day. An increase in the number of litters exhibiting unossification of sternebrae #5 and/or #6 was noted exclusively at 500 mg/kg/day and was correlated with fetal body weight reduction. Maternal appearance and behavior and the incidence of fetal malformations were not affected at any dosage level. Based on these results, Santicizer 9 did not produce a teratogenic effect when administered orally to mated Charles River COBS CD rats at 500 mg/kg/day and less.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High. There is one recent GLP full OECD 414 comparable study available in rats showing no teratogenic effects at toxic dose levels of TSA (o,p -Toluene sulphonamide) and additionally one recent GLP full OECD 414 study available in rabbits, and information from an OECD 422 screening study in rat which included evaluations for skeletal malformations.
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

1333 -07 -9,Developmental toxicity / teratogenicity, Int. R&D Corp., 401 -290,1985, RS-copy

There is one GLP compliant study on o/p-TSA available that is comparable to an OECD 414 study available in rats.

 

o/p-TSA was administered orally by gavage to groups of 25 mated rats per dose level of 0 (vehicle only, corn oil), 50, 250 and 500 mg/kg/day on days 6 through 15 of gestation. Cesarean sections were performed on all surviving females on gestation day 20 and the foetuses were removed for teratologic evaluation.

Survival was 100% among all groups on study. At 250 and 500 mg effects were observed in maternal body weight gain and several Cesarean section parameters. Statistically significant reductions were also noted at 500 mg/kg/day during the overall gestation period. Foetotoxicity was apparent at 250 and 500 mg/kg/day as evidenced by increased postimplantation loss which was statistically significant at 500 mg/kg/day. In addition, a treatment-related trend of foetal body weight inhibition was noted at all dosage levels and values were significantly different from the control, p<0.01 at 250 and 500 mg/kg/day. An increase in the number of litters exhibiting delayed ossification was noted exclusively at 500 mg/kg/day and was correlated with foetal body weight reduction. Maternal appearance and behaviour and the incidence of foetal malformations were not affected at any dosage level. Based on these results, o/p-TSA did not produce a teratogenic effect when administered orally to mated rats at 500 mg/kg/day and less. A NOAEL of 50 mg/kg bw/d was established for maternal and developmental toxicity.

Cross-reading (RA) between Toluenesulphonamides (o-TSA, p-TSA and o/p-TSA) is justified and already previously described byHouthoff, E. andGyimesi, M. (2013) and was submitted to ECHA in the dossier of o/p-TSA and this dossier. The available data confirm the comparable hazard profiles for p-TSA and o-TSA covering all physical-chemical ecotoxicological en toxicological endpoints. Further support is provided by the available data on o/p-TSA. As this is basically a mixture of p-TSA and o-TSA its study results can be expected to reflect the same hazard profile.

70-55-3, Developmental toxicity / teratogenicity, Notox, 474097, 2008

p-TSA was evaluated in a Prenatal Developmental Toxicity Study following OECD 414 guidelines under GLP. Four groups of twenty-four New Zealand White rabbits were inseminated (Day 0post-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 29post-coitum. The study consists of Part I and II. Dietary concentrations were analytically verified.

Possibly stress from construction work interfered with the performance of the study, resulting to very low pregnancy rates, including in the controls, which required additional groups of animals (part II) to be dosed. In the study report the data is reported as one pooled group as well as separately for each cohort.

 

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 during the first days of dosing, but this is not considered as adverse effect in this evaluation. 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 foetal morphology occurred at dose levels of 1000 and 3000 ppm.

Therefore, a dosage level of 3000 ppm (113 mg/kg body weight/day in rabbits) was considered to be the No Observed Adverse Effect Level (NOAEL) for embryo/foetal 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).

 

70-55-3, Toxicity to reproduction, MHW Japan, 1994

(Reported under toxicity for reproduction) Additionally there is a study screening for reproduction toxicity according to OECD 422 Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test in rat by gavage under GLP.

This study included evaluation of offspring for visceral and skeletal malformation indicating no teratogenic effects of 4-methylbenzenesulfonamide.

 

70-55-3, Toxicity to reproduction 2-gen rat, NOTOX 474064, 2007

(Reported under toxicity for reproduction) The available full two-generation study according to OECD 416 in rats by dietary admixture concluded to a developmental toxicity related to decreased body weights of pups at 10000 ppm, accompanied by organ weight changes for the brain, spleen and thymus and a delay in vaginal opening and balano-preputial separation.Consequently, the NOAEL for developmental toxicity was established at 3000 ppm (corresponding to165-237 mg/kg bw/day for males and 232-499 mg/kg bw/day for females).

 Justification for selection of Effect on developmental toxicity: via oral route: Only available high quality study evaluating developmental toxicity

 

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

Only available high quality study evaluating developmental toxicity

 

 

Justification for classification or non-classification

Toxicity to reproduction has been sufficiently evaluated in studies addressing both fertility and foetal development. Available studies for the evaluation of of p-TSA for these endponts indicated no concerns with respect to hazards related to reproduction, and thus no classification is required.

Based on the low lipophilic (Pow = 0.6) aspect of the substance, the quick absorption and subsequent excretion in urine, accumulation of high levels into breast milk is not likely. Also the 2-generation study shows no indication for a concern for effects on or via lactation.