Calcium bis[4-[[1-[[(2-methylphenyl)amino]carbonyl]-2-oxopropyl]azo]-3-nitrobenzenesulphonate]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive/Developmental Toxicity Screening Study: NOAEL (fertility/develompent) >= 1 100 mg/kg bw/day, according to OECD 422, GLP-compliant, rat, 2012, K1

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2012 - 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

22 Mar 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

Limit test:

no

Specific details on test material used for the study:

solid yellow

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): water
- Storage temperature of food: RT

- applied as a suspension

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): pregnant animals and litter together

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- according GLP
- stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study
- method stability of test item in drinking water: UV/VIS spectroscopy

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males,
and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females.

Frequency of treatment:

daily

Details on study schedule:

- Age at mating of the mated animals in the study: 13-14 weeks

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 110 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: range finder test 300 and 1000 mg/kg bw, no effects up to 1000 mg/kg bw
- Rationale for animal assignment (if not random): Randomization

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).

FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose

URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sperm parameters (parental animals):

stages of spermatogenesis were examined in histopathology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality up to day 4, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina

HISTOPATHOLOGY
ORGAN WEIGHTS: Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

HISTOPATHOLOGY: Yes, control and high dose group, gross lesions in all animals
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Oviducts
20. Peyer’s patches
21. Prostate
22. Rectum
23. Sciatic nerve
24. Seminal vesicles
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Spleen
27. Stomach (forestomach and glandular stomach)
28. Testes
29. Thymus
30. Thyroid glands
31. Trachea
32. Urinary bladder
33. Uterus
34. Vagina

Postmortem examinations (offspring):

SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All pups delivered from the F0 parents were examined as soon as possible on the day of birth

GROSS NECROPSY
All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.

Statistics:

Blood parameters:
For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose
group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Urinalysis parameters: WILCOXON-test (one-sided)

Food consumption: DUNNETT-test (twosided)

fertility indices: FISHER'S EXACT test

Proportions of affected pups per litter with necropsy observations: WILCOXON-test

Weight parameters: KRUSKAL-WALLIS test

Reproductive indices:

Male reproduction data:
- Male mating index
- Male fertility index

Female reproduction and delivery data:
- Female mating index
- Female fertility index
- Gestation index
- Live birth index
- Post implantation loss

Offspring viability indices:

Pup number and status at delivery
Pup viability/mortality
Sex ratio
Pup body weight data

Clinical signs:

no effects observed

Description (incidence and severity):

All male and female animals of test group 2 (330 mg/kg bw/d) and 3 (1110 mg/kg bw/d) showed yellowish discolored feces from day 7 until the end of the entire study during premating, mating and postmating period.
All female animals of test group 2 (330 mg/kg bw/d) and 3 (1110 mg/kg bw/d) showed yellowish discolored feces during the whole gestation period.
One female animal (No. 113) of test group 1 (110 mg/kg bw/d) showed palpable mass through skin from gestation day 12 until the end of the period. This finding was assessed as being spontaneous in nature.
One sperm-positive F0 female of the test group 1 (No. 119) and 1 sperm-negative F0 female of the test group 3 (No. 137) did not deliver any F1 pups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and mean body weight gain of the F0 males in test groups 1-3 (110, 330 and 1110 mg/kg bw/d) were comparable to the concurrent control throughout the entire study period.
Mean body weight and mean body weight gain of the F0 females in test groups 1-3 (110, 330 and 1110 mg/kg bw/d) were comparable to the concurrent control throughout the entire premating, gestation and lactation periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (110, 330 and 1110 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, covering premating, gestation and lactation.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.
In males of test group 2 (300 mg/kg bw/d), red blood cell (RBC) counts were lower compared to controls, but this decrease was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were observed.
In females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) glucose levels were lower compared to controls. This was the only changed parameter in these animals and therefore, the alteration was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among urinalysis parameters were observed.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The discoloration of the content in the digestive tract was regarded to be a consequence to the oral intake of the test substance which is of yellow color. Therefore, the gross findings in the remaining animals were not investigated in addition.
Only in some animals the gross findings “discoloration of contents” could be approved by light microscopy. This was regarded to be due to clearing of the stomach and intestine before histotechnical processing.
All other findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.

Other effects:

no effects observed

Description (incidence and severity):

Functional observational battery (Home cage observations, Open field observations, Sensorimotor tests/reflexes, Quantitative Parameters, Motor activity measurement)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The male mating index was 100% in test groups 0, 1 and 2 and 90% in test group 3. This finding reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter.
One male of test group 1 (No. 19 mated with female No. 119) and 1 male of test group 3 (No. 37 mated with female No. 137) did not generate F1 pups.
The male fertility index was 100% in test group 0 and 2 and 90% in test group 1 and 3.
The female mating index calculated after the mating period for F1 litter was 100% in test groups 0, 1 and 2 and 90% in test group 3. The mean duration until sperm was detected (GD 0) was 2.3, 2.1, 2.0 and 2.2 days in test groups 0-3. This finding reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
All sperm positive rats delivered pups with the exception of female No. 119 (test group 1), which was mated with male No. 19 and did not become pregnant.
Female animal No. 137 (test group 3) which was mated with male No. 37 did not get sperm in vaginal smear.
The female fertility index was 100% in test group 0, 2 and 3 and 90% in test group 1. Female animals Nos. 119 and 137, which delivered no pups, showed no implantation sites.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The viability index indicating pup mortality during lactation (PND 0 - 4) was 98% in the control, 100% (test group 1), 95 % (test group 2) and 99% (test group 3) and was in the normal range of biological variation inherent in the strain of rats used for this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
Two male runts were seen in control group. One female runt was seen in test group 2 (330 mg/kg bw/d).

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

One F1 pup of test group 1 (110 mg/kg bw/d) showed discolored liver, one pup of test group 3 (1110 mg/kg bw/d) showed situs inversus. One pup of control group and one pup of test group 2 (330 mg/kg bw/d) were cannibalized.
These findings were assessed as being spontaneous in nature and without biological relevance.

Histopathological findings:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 110 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 3: Significantly altered absolute organ weights

	Male animals
Test group (mg/kg bw/day)	1 (110)	2 (330)	3 (1110)
Brain	99%	95%**	101%

* p ≤ 0.05, ** p ≤ 0.01

Table 4: Significantly altered relative organ weights

	Male animals
Test group (mg/kg bw/day)	1 (110)	2 (330)	3 (1110)
Heart	86%**	87%**	89%*

* p ≤ 0.05, ** p ≤ 0.01

Table 5: Relevant gross findings

	Male animals				Female animals
Test group (mg/kg bw/day)	0 (0)	1 (110)	2 (330)	3 (1110)	0 (0)	1 (110)	2 (330)	3 (1110)
No of animals	10	10	10	10	10	10	10	10
Glandular stomach Discoloration of contents		5	7	10		1	4	8
Jejunum Discoloration of contents			2	9			1	2
Cecum Discoloration of contents								2
Colon Discoloration of contents			2	9				3

Table 6: Male fertility indices for F0 males

	Test group 0 (0 mg/kg bw/d)	Test group 1 (110 mg/kg bw/d)	Test group 2 (330 mg/kg bw/d)	Test group 3 (1110 mg/kg bw/d)
Male fertility index [%]	100	90	100	90

* p ≤ 0.05; ** p ≤ 0.01

 

Table 7: Fertility indices for F0 females

	Test group 0 (0 mg/kg bw/d)	Test group 1 (110 mg/kg bw/d)	Test group 2 (330 mg/kg bw/d)	Test group 3 (1110 mg/kg bw/d)
Female fertility index [%]	100	90	100	100

* p ≤ 0.05; ** p ≤ 0.01

 

Table 8: Sex ratio of live F1 pups

PND 0	Test group 0 (0 mg/kg bw/d)	Test group 1 (110 mg/kg bw/d)	Test group 2 (330 mg/kg bw/d)	Test group 3 (1110 mg/kg bw/d)
Live males [%]	55.1	55.1	44.1	55.8
Live females [%]	44.9	44.9	55.9	44.2
PND 4
Live males [%]	55.2	55.1	44.6	56.3
Live females [%]	44.8	44.9	55.4	43.7

 

Table 9: Number of males with no offspring and of not pregnant females

	Male animals				Female animals
Test group (mg/kg bw/day)	0 (0)	1 (110)	2 (330)	3 (1110)	0 (0)	1 (110)	2 (330)	3 (1110)
Number of animals	10	10	10	10	10	10	10	10
No offspring		1		1
Animal not pregnant						1		1

Conclusions:

Thus, under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test the oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1110 mg/kg bw/d in both sexes. The NOAEL for reproductive performance, fertility and developmental toxicity was set to 1110 mg/kg bw/d in male and female Wistar rats.

Executive summary:

The test substance was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/d (test group 0), 110 mg/kg bw/d (test group 1), 330 mg/kg bw/d (test group 2) and 1110 mg/kg bw/d (test group 3). Regarding clinical examinations, no signs of general systemic toxicity were observed in male or female parental animals of all test groups (110, 330 and 1110 mg/kg bw/d) during the entire study.
The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and two weeks thereafter in females.

A detailed clinical observation (DCO) was performed weakly in all animals. Food consumption and body weights of F0 parents were monitored throughout the study. Towards the end of the administration period clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group. Additionally, a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The pups were sexed and examined for macroscopically evident changes. They were weighed and their viability was recorded. At necropsy, all pups were sacrificed examined macroscopically for external and visceral findings.

No treatment-related, adverse effects were observed up to a dose of 1110 mg/kg bw/d regarding clinical pathology, fertility and reproductive performance.

Regarding pathology, macroscopically yellow discoloration of the content of the digestive tract in numerous animals was observed. In single treated animals and single organs of the digestive tract these yellow pigments could also be observed histopathologically. Beside the discoloration no signs of toxicity in the respective tissues were noted. This finding is regarded to be a consequence to the oral intake of the yellow test substance and therefore treatment related but not adverse in nature.
The treatment of male and female Wistar rats with up to 1110 mg/kg bw of the test substance did not lead to adverse findings. Especially no effects of the test substance on the reproduction tract were observed. All other findings were considered to be incidental or spontaneous in origin and without
any relation to treatment.

Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 1110 mg/kg bw/d.

The NOAEL for developmental toxicity in the F1 progeny was found to be 1110 mg/kg bw/d.

The NOAEL for general, systemic toxicity was 1110 mg/kg bw/d.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 110 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive/Developmental Toxicity Screening Study:

The test substance was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/d (test group 0), 110 mg/kg bw/d (test group 1), 330 mg/kg bw/d (test group 2) and 1110 mg/kg bw/d (test group 3). Regarding clinical examinations, no signs of general systemic toxicity were observed in male or female parental animals of all test groups (110, 330 and 1110 mg/kg bw/d) during the entire study.
The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and two weeks thereafter in females.

A detailed clinical observation (DCO) was performed weakly in all animals. Food consumption and body weights of F0 parents were monitored throughout the study. Towards the end of the administration period clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group. Additionally, a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The pups were sexed and examined for macroscopically evident changes. They were weighed and their viability was recorded. At necropsy, all pups were sacrificed examined macroscopically for external and visceral findings.

No treatment-related, adverse effects were observed up to a dose of 1110 mg/kg bw/d regarding clinical pathology, pathology, fertility and reproductive performance.

Effects on developmental toxicity

Description of key information

Reproductive/Developmental Toxicity Screening Study: NOAEL (fertility/develompent) >= 1 100 mg/kg bw/day, according to OECD 422, GLP-compliant, rat, 2012, K1

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2012 - 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 422

Version / remarks:

22 Mar 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

solid yellow

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 9th, 2011 To:October 6th, 2011

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): water
- Storage temperature of food: RT

- applied as a suspension

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- according GLP
- stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study
- method stability of test item in drinking water: UV/VIS spectroscopy

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for maximum 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males,
and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 110 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: range finder test 300 and 1000 mg/kg bw, no effects up to 1000 mg/kg bw
- Rationale for animal assignment (if not random): Randomization

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).

FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose

URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

- External examinations: Yes, all pubs
- Soft tissue examinations: necropsy observations macroscopically
- Skeletal examinations: No
- Head examinations: No
- Sex ratio
- Pup body weight data
- Pup clinical observations

Statistics:

Blood parameters:
For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose
group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Urinalysis parameters: WILCOXON-test (one-sided)

Food consumption: DUNNETT-test (twosided)

fertility indices: FISHER'S EXACT test

Proportions of affected pups per litter with necropsy observations: WILCOXON-test

Weight parameters: KRUSKAL-WALLIS test

Indices:

Pup number and status at delivery
Pup viability/mortality
Sex ratio
Pup body weight data

Clinical signs:

no effects observed

Description (incidence and severity):

All male and female animals of test group 2 (330 mg/kg bw/d) and 3 (1110 mg/kg bw/d) showed yellowish discolored feces from day 7 until the end of the entire study during premating, mating and postmating period.
All female animals of test group 2 (330 mg/kg bw/d) and 3 (1110 mg/kg bw/d) showed yellowish discolored feces during the whole gestation period.
One female animal (No. 113) of test group 1 (110 mg/kg bw/d) showed palpable mass through skin from gestation day 12 until the end of the period. This finding was assessed as being spontaneous in nature.
One sperm-positive F0 female of the test group 1 (No. 119) and 1 sperm-negative F0 female of the test group 3 (No. 137) did not deliver any F1 pups.
All female animals of test group 2 (330 mg/kg bw/d) and 3 (1110 mg/kg bw/d) showed yellowish discolored feces during the entire lactation period.
One female animal (No. 113) of test group 1 (110 mg/kg bw/d) showed palpable mass through skin during the whole lactation period. This finding was assessed as being spontaneous in nature.

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight and mean body weight gain of the F0 females in test groups 1-3 (110, 330 and 1110 mg/kg bw/d) were comparable to the concurrent control throughout the entire premating, gestation and lactation periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (110, 330 and 1110 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, covering premating, gestation and lactation.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were observed.
In females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) glucose levels were lower compared to controls. This was the only changed parameter in these animals and therefore, the alteration was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among urinalysis parameters were observed.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The weight decrease in absolute brain weight in males of test group 2 (330 mg/kg bw/day) was regarded to be incidental due to a missing dose response relationship and missing histopathologic findings in test group 3 (1110 mg/kg bw). The decrease in heart weights of males in all test groups was also regarded to be incidental as no histopathologic correlate could explain the weight decrease.
All other mean weight parameters did not show significant differences when compared to the control groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Several animals of test group 1, 2 and 3 revealed a yellow discoloration of the contents of the glandular stomach, small and large intestines. These findings are regarded to be treatment related.
All other gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The discoloration of the content in the digestive tract was regarded to be a consequence to the oral intake of the test substance which is of yellow color. Therefore, the gross findings in the remaining animals were not investigated in addition.
Only in some animals the gross findings “discoloration of contents” could be approved by light microscopy. This was regarded to be due to clearing of the stomach and intestine before histotechnical processing.
All other findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.

Other effects:

no effects observed

Description (incidence and severity):

Functional observational battery (Home cage observations, Open field observations, Sensorimotor tests/reflexes, Quantitative Parameters, Motor activity measurement)

Number of abortions:

no effects observed

Description (incidence and severity):

The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 97.5% and 99% for test group 0 and 2, respectively. For test groups 1 and 3, the live birth indices were 100%.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The postimplantation loss in test group was 8.8% in test group 0, 8.9% in test group 1, 7.8% in test group 2 and 5.6% in test group 3. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Details on maternal toxic effects:

Regarding pathology, macroscopically yellow discoloration of the content of the digestive tract in numerous animals was observed. In single treated animals and single organs of the digestive tract these yellow pigments could also be observed histopathologically. Beside the discoloration no signs of toxicity in the respective tissues were noted. This finding is regarded to be a consequence to the oral intake of the yellow test substance and therefore treatment related but not adverse in nature.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 110 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
Two male runts were seen in control group. One female runt was seen in test group 2 (330 mg/kg bw/d).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The viability index indicating pup mortality during lactation (PND 0 - 4) was 98% in the control, 100% (test group 1), 95 % (test group 2) and 99% (test group 3) and was in the normal range of biological variation inherent in the strain of rats used for this study.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 110 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 3: Significantly altered absolute organ weights

	Male animals
Test group (mg/kg bw/day)	1 (110)	2 (330)	3 (1110)
Brain	99%	95%**	101%

* p ≤ 0.05, ** p ≤ 0.01

Table 4: Significantly altered relative organ weights

	Male animals
Test group (mg/kg bw/day)	1 (110)	2 (330)	3 (1110)
Heart	86%**	87%**	89%*

* p ≤ 0.05, ** p ≤ 0.01

Table 5: Relevant gross findings

	Male animals				Female animals
Test group (mg/kg bw/day)	0 (0)	1 (110)	2 (330)	3 (1110)	0 (0)	1 (110)	2 (330)	3 (1110)
No of animals	10	10	10	10	10	10	10	10
Glandular stomach Discoloration of contents		5	7	10		1	4	8
Jejunum Discoloration of contents			2	9			1	2
Cecum Discoloration of contents								2
Colon Discoloration of contents			2	9				3

Table 6: Male fertility indices for F0 males

	Test group 0 (0 mg/kg bw/d)	Test group 1 (110 mg/kg bw/d)	Test group 2 (330 mg/kg bw/d)	Test group 3 (1110 mg/kg bw/d)
Male fertility index [%]	100	90	100	90

* p ≤ 0.05; ** p ≤ 0.01

 

Table 7: Fertility indices for F0 females

	Test group 0 (0 mg/kg bw/d)	Test group 1 (110 mg/kg bw/d)	Test group 2 (330 mg/kg bw/d)	Test group 3 (1110 mg/kg bw/d)
Female fertility index [%]	100	90	100	100

* p ≤ 0.05; ** p ≤ 0.01

 

Table 8: Sex ratio of live F1 pups

PND 0	Test group 0 (0 mg/kg bw/d)	Test group 1 (110 mg/kg bw/d)	Test group 2 (330 mg/kg bw/d)	Test group 3 (1110 mg/kg bw/d)
Live males [%]	55.1	55.1	44.1	55.8
Live females [%]	44.9	44.9	55.9	44.2
PND 4
Live males [%]	55.2	55.1	44.6	56.3
Live females [%]	44.8	44.9	55.4	43.7

 

Table 9: Number of males with no offspring and of not pregnant females

	Male animals				Female animals
Test group (mg/kg bw/day)	0 (0)	1 (110)	2 (330)	3 (1110)	0 (0)	1 (110)	2 (330)	3 (1110)
Number of animals	10	10	10	10	10	10	10	10
No offspring		1		1
Animal not pregnant						1		1

Conclusions:

Thus, under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. The NOAEL for reproductive performance, fertility and developmental toxicity was set to 1110 mg/kg bw/d in male and female Wistar rats.

Executive summary:

The test substance was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/d (test group 0), 110 mg/kg bw/d (test group 1), 330 mg/kg bw/d (test group 2) and 1110 mg/kg bw/d (test group 3). Regarding clinical examinations, no signs of general systemic toxicity were observed in male or female parental animals of all test groups (110, 330 and 1110 mg/kg bw/d) during the entire study.
The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and two weeks thereafter in females.

A detailed clinical observation (DCO) was performed weakly in all animals. Food consumption and body weights of F0 parents were monitored throughout the study. Towards the end of the administration period clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group. Additionally, a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The pups were sexed and examined for macroscopically evident changes. They were weighed and their viability was recorded. At necropsy, all pups were sacrificed examined macroscopically for external and visceral findings.

No treatment-related, adverse effects were observed up to a dose of 1110 mg/kg bw/d regarding clinical pathology, fertility and reproductive performance.

Regarding pathology, macroscopically yellow discoloration of the content of the digestive tract in numerous animals was observed. In single treated animals and single organs of the digestive tract these yellow pigments could also be observed histopathologically. Beside the discoloration no signs of toxicity in the respective tissues were noted. This finding is regarded to be a consequence to the oral intake of the yellow test substance and therefore treatment related but not adverse in nature.
The treatment of male and female Wistar rats with up to 1110 mg/kg bw of the test substance did not lead to adverse findings. Especially no effects of the test substance on the reproduction tract were observed. All other findings were considered to be incidental or spontaneous in origin and without
any relation to treatment.

Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 1110 mg/kg bw/d.

The NOAEL for developmental toxicity in the F1 progeny was found to be 1110 mg/kg bw/d.

The NOAEL for general, systemic toxicity was 1110 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 110 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Reproductive/Developmental Toxicity Screening Study:

The test substance was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/d (test group 0), 110 mg/kg bw/d (test group 1), 330 mg/kg bw/d (test group 2) and 1110 mg/kg bw/d (test group 3). Regarding clinical examinations, no signs of general systemic toxicity were observed in male or female parental animals of all test groups (110, 330 and 1110 mg/kg bw/d) during the entire study.
The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and two weeks thereafter in females.

A detailed clinical observation (DCO) was performed weakly in all animals. Food consumption and body weights of F0 parents were monitored throughout the study. Towards the end of the administration period clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group. Additionally, a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The pups were sexed and examined for macroscopically evident changes. They were weighed and their viability was recorded. At necropsy, all pups were sacrificed examined macroscopically for external and visceral findings.

No treatment-related, adverse effects were observed up to a dose of 1110 mg/kg bw/d regarding clinical pathology, pathology, fertility and reproductive performance.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available screening study is reliable and suitable for classification purposes under Regulation 1272/2008. No adverse effects were observed in a screening study regarding fertility and developmental toxicity (OECD 422). As a result, the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the fifteenth time in Regulation (EC) No. 2020/1182.

During the four days covered in the screening study, no effects via lactation were observed.