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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study according to guideline. Although only summary available it has been peer internationally reviewed within OECD and assigned reliability 1.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tosyl chloride
EC Number:
202-684-8
EC Name:
Tosyl chloride
Cas Number:
98-59-9
Molecular formula:
C7H7ClO2S
IUPAC Name:
4-methylbenzenesulfonyl chloride
Details on test material:
4-Methylbenzenesulfonyl chloride, purity = 99.7 %,
Fluka, Lot No. - 422308/1

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
- male/female
- Age of animals at study: 9 weeks old for males and females
- Weight at study repeated dose toxicity: 325.8 - 363.1 g for males and 198.5 - 229.3 g for females
- Number of test animals: 60 animals for each sex

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
not specified
Details on mating procedure:
Each male and female rat was selected from the same test group in order to copulate. It spent 14 days in terms of mating. The day after the copulating, mating would be determined through the observation of sperm in a vaginal rinse.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Exposure period : 34, 36 - 45, and 51 days for male, copulated female and not copulated female animals, respectively.
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 150, 350 and 750 mg/kg/day
Basis:

No. of animals per sex per dose:
12 animals/sex/dose, plus 6 in 14-day recovery group/sex for control and high dose
Control animals:
yes, concurrent no treatment
Details on study design:
According to the preliminary test (Report No. P104), dose level was set to 0, 150, 350 and 750 mg/kg/day.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily; mortality: twice/day
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: once a week and just before the necropsy, but in case of pregnant females, it was measured on the day 0, 7, 14, 20 of the gestation period, date of delivery, and 4 days of the lactation day.

FOOD CONSUMPTION:
- Food consumption: Yes: once a week except mating period

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Not determined.
Sperm parameters (parental animals):
Not determined.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- The number of survivors and deaths during delivery
- Body weight and Survival rate: measured on the day of delivery and on the day 4 of lactation.
- Sex ratio: It is determined by anogenital distance, for more than 2 mm and less than 1 mm were male and female, respectively.
- Examination of the external surface of pups: It was observed on the day of delivery and on the day 4 of lactation to determine abnormalities.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes

ORGAN WEIGHT: testes, epididymider (all males) liver, kidney, adrenals, thymus, spleen, brain and heart (5 male and female rats from each test group).

HISTOPATHOLOGY: Yes
22 tissues were fixed to perform histopathological evaluations including: testes, epididymides, ovaries, accessory sex organs for all animals, brain (including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer’s patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea, lungs, uterus, urinary bladder, lymph nodes (cervical mesenteric), peripheral nerve (sciatic or tibial), and bone marrow.
Postmortem examinations (offspring):
No infoirmation
Statistics:
Statistical decision tree, but in case of recovery group, either two-side Student’s t-test or two-side Aspin-Welch t-test was used. In case of categorical data, two-sided Fisher’s exact test was used.
Reproductive indices:
- Copulation index
- number of implantations
- number of corpus luteum
- Number of females pregnant
Offspring viability indices:
- Number of life pups
- post natal loss
- pre and post-implantation loss

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
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:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality males; in females 350 mg/kg: 2/12 and at 750 mg/kg: 4/18
In the control groups, there were no specific clinical symptoms during test period.
150 mg: Intermittent (blood-like) salivation and staining around mouth (3/18 males, 6/18 females), and in females at delivery: difficult delivery, poor nursing, irregular respiration, uterus introsusception and piloerection.
350 mg: Intermittent (blood-like) salivation and staining around mouth were observed after day 15 and (blood-like) staining around nose (7/12 males, 4/12 females). Iintermittent soft stool and staining around anorectal region were observed for the most male animals and in 2 females. One female showed difficult delivery, lacrimation, and irregular respiration from day 4.
750 mg: soft stool and staining around anorectal region for most males and all females; and 5 cases of loss of hair around tail region were observed. Intermittent (blood-like) salivation and 9 cases of staining around mouth and 9 cases of (blood-like) staining around nose. Soft stool and staining around anorectal region for all animals; some cases of intermittent diarrhea were observed. Some animals with found dead and in dying condition had symptoms such as irregular respiration, crawing position, hypoactivity, and abdominal swelling.
In the 750 mg/kg/day recovery group, salivation, staining around mouth, soft stool and staining around anorectal region were not observed during the recovery period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
From start of dosing BW gain at 750 mg/kg group a bit lower resulting to a lower BW of 8% in males and 5.5% in females compared to controls during first week of recovery which partly recovered during the second week of the recovery period.
Only during the first week food consumption some somewhat lower in the 705 mg group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Pregnancy and delivery: No effects were seen between the groups in duration of pregnancy, number of corpus luteum, number of implantation, pre-implantation loss, post-implantation loss
- Index of copulation, fertility and gestation: There was no significant difference between the control and treatment group in terms of copulation, fertility and gestation index. The sex mis-confirmation for new born pups in this test did not have relationship with test substance since its frequency was low and no dose-correlation. (See Table)

ORGAN WEIGHTS (PARENTAL ANIMALS)
No effects were seen in sex-organ weights. Weight of the spleen was increased in treatment groups compared to controls for both males and females, although in females not relative weight. No differences in spleen weights were seen in recovery groups. In males the absolute, but not the relative, weight of the heart was decreased in the 750 mg/kg group. In the female recovery group the weights for adrenal and the brain were increased. (See table)

GROSS PATHOLOGY (PARENTAL ANIMALS)
No information provided

HISTOPATHOLOGY (PARENTAL ANIMALS)
See table.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL parental: Above the highest tested dose (750 mg/kg/day)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Examination of the external surface of pups
Histopathological findings:
not specified

Details on results (F1)

Number of pups born, viability and sex ratio: No significant difference was observed between the treatment groups and the control group at the time of the delivery and on the day 4 of the lactation.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Mating, fertility, gestation and viability data:

DOSE: (mg/kg) 

0

150

350

750

 No. of mated males 

12

12

12

11

 Copulation index (%) 

100

100

100

90.9

 Fertility index (%) 

91.7

100

100

100

 No. of mated females 

12

12

12

11

 Copulation index (%) 

100

100

100

90.9

 Fertility index (%) 

91.7

100

100

100

 Gestation index (%) 

100

91.7

83.3

90

 No. of corpora lutea 

14.9

14.5

15.1

14.1

 Mean±S.D

1.1

2.5

1.7

0.9

 No. of implantations

14.4

12.7

12.7

13.3

 Mean±S.D

1

4.2

2.8

1

 Mean % preimplantation loss

3.5

15.5

14.3

5.4

 No. of embryo/fetal death 

2

1.7

2

2.3

 No. of live pups born 

12.4

12

10.7

11

 Mean±S.D

0.9

2.5

3.3

3.2

 Mean pregnancy period (day) 

21.7

21.8

21.8

21.9

 Viability at birth pp 

95.4

92.1

93.9

92.3

 Viability at LD 4 

99.2

97.8

88.3

99

 Body weights of pups (g) 

 

 

 

 

 Male (at birth)

6.25

5.99

6.37

5.98

 Day 4 

8.57

8.56

9.71

8.7

 Female (at birth)

5.75

5.62

5.73

5.59

 Day 4 

8.03

7.98

8.59

8.21

 

 

Organ weights:

Dose (mg/kg) 

0

150

350

750

 satellite 

0

750

Absolute (sex) organ weight of males

Testes(g) 

3.04

3.285

3.332

3.203

3.337

3.208

Epididymis(g) 

1.258

1.29

1.37

1.3

1.388

1.335

Liver (g) 

10.698

11.142

10.698

10.01

11.139

10.736

Thymus(g) 

0.355

0.307

0.356

0.32

0.31

0.297

Kidneys(g) 

2.364

2.272

2.502

2.385

2.397

2.474

Adrenals(g) 

0.061

0.064

0.062

0.068

0.053

0.055

Spleen(g) 

0.632

0.725

0.783

0.794

0.844

0.716

Brain(g) 

1.985

1.947

2.022

1.91

2.064

2.018

Heart(g) 

1.325

1.244

1.282

1.182

1.411

1.382

Absolute organ weight of females

Liver (g) 

6.947

6.886

7.298

7.431

6.855

6.621

Kidneys(g) 

1.46

1.451

1.433

1.5

1.523

1.509

Adrenals(g) 

0.071

0.073

0.073

0.072

0.057

0.066

Thymus(g) 

0.106

0.14

0.161

0.096

0.298

0.291

Brain(g) 

1.884

1.885

1.883

1.855

1.742

1.87

Spleen(g) 

0.397

0.419

0.494

0.476

0.477

0.472

Heart(g) 

0.818

0.809

0.853

0.767

0.876

0.829

 

Histopathology:

Histopathological findings of males (Group)

 Dose level (mg/kg/day) 

0

150

350

700

 No. of animals examined 

5

5

5

5

 Observation(s) 

 No. of animals observed 

No significant findings 

2

4

4

3

Liver: focal necrosis

 

 

 

 

Minimal 

0

0

0

1

Nongrandular stomach: epithelial proliferation and vacuolation

 

 

 

 

Minimal

0

2

0

0

Moderate

0

3

0

0

Marked 

0

0

5

5

Nongrandular stomach: hyperkeratosis

 

 

 

 

Minimal 

0

3

0

0

Slight 

0

2

5

5

Nongrandular stomach: submucosal edema

 

 

 

 

Minimal 

0

0

0

1

Slight 

0

0

4

4

Moderate

0

3

1

0

Nongrandular stomach: inflammation

 

 

 

 

Slight 

0

3

5

5

No. of animals examined (reproductive organs)

12

12

12

12

Observation(s) 

 No. of animals observed 

No significant findings

10

12

12

10

Testes: atrophy and degeneration of seminiferous tubules

 

 

 

 

Minimal

0

0

0

1

Moderate

2

0

0

0

Marked 

0

0

0

1

Epididymides: oligospermia

 

 

 

 

Minimal

1

0

0

0

Marked 

1

0

0

1

 

Histopathological findings of females (Group)

 Dose level (mg/kg/day) 

0

150

350

700

 No. of animals examined 

5

0

0

0

 Observation(s) 

 No. of animals observed 

No significant findings 

5

1

0

0

Liver: focal necrosis

 

 

 

 

Minimal 

0

0

0

0

Heart: inflammatorycell foci

 

 

 

 

Minimal 

1

0

0

0

Nongrandular stomach: epithelial proliferation and vacuolation

 

 

 

 

Minimal

0

1

1

1

Slight 

0

2

3

3

Marked 

0

0

1

1

Nongrandular stomach: hyperkeratosis

 

 

 

 

Minimal 

0

1

3

1

Slight 

0

0

2

3

Moderate

0

0

0

1

Nongrandular stomach: submucosal edema

 

 

 

 

Slight 

0

1

1

1

Nongrandular stomach: inflammation

 

 

 

 

Minimal 

0

1

2

3

No. of animals examined (reproductive organs)

12

12

10

9

Observation(s) 

 No. of animals observed 

No significant findings

12

12

10

9

 

Applicant's summary and conclusion

Conclusions:
There were no reproductive effects seen at 750 mg/kg, the highest dose tested
Executive summary:

Tosyl chloride has been evaluated in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD 422 and in compliance to GLP.

Tosyl chloride was dose by gavage to groups of 12 Sprague-Dawley rats/sex/dose level at levels of 0, 150, 350 and 750 mg/kg/day for 35 days and 36 - 51 days for male and female rats, respectively. A separate group of 6 animals/sex were added for a 14-day recovery group to the control and the high dose groups.

Results:

Parental toxicity: No mortality males; in females 350 mg/kg: 2/12 and at 750 mg/kg: 4/18.

Some clinical signs were observed at the dose level of 150 mg/kg/day in both male and females such as intermittent (blood-like) salivation and staining around mouth. Effects were more severe at 350 mg/kg and at 750 mg/kg reported clinical signs were soft stool and staining around anorectal region for most males and all females; and 5 cases of loss of hair around tail region were observed. Intermittent (blood-like) salivation and 9 cases of staining around mouth and 9 cases of (blood-like) staining around nose. Soft stool and staining around anorectal region for all animals; some cases of intermittent diarrhea were observed. A decreased BW gain was observed in the 750 mg group resulting to a lower BW that was maximal 8% lower in males and 5.5% in females compared to controls. Food consumption was a bit lower during the first week in the 750 mg group. Histopathology showed dose related effects of epithelial proliferation and inflammation of the forestomach already visible in animals treated at 150 mg.

There were no further major changes observed in other parameters (haematology, biochemistry, neurobehavioral functions, organ weights)

In the 750 mg/kg/day recovery group, salivation, staining around mouth, soft stool and staining around anorectal region were not observed during the recovery period.

 

Reproductive parameters were not affected.No effects were seen between the groups in duration of pregnancy, number of corpus luteum, number of implantation, pre-implantation loss, post-implantation loss. There was no significant difference between the control and treatment group in terms of copulation, fertility and gestation index.

Among the clinical signs was remarked for the 150 mg/kg treated group only that thefemale animals additionally showed difficulty in delivery, poor nursing, and irregular respiration. However, as this is not remarked for the higher dose groups, and no effects are seen in any of the reproductive parameters, these likely represents an accidental observation.

 

Consequently the NOAEL for fertility was established at 750 mg/kg bw/day, the highest dose tested.

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