[1-[[(2-hydroxyphenyl)imino]methyl]-2-naphtholato(2-)-N,O,O']copper

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014 - 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

Dose-Range-Finder

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 (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 220 g (females) and 310g (males)
- 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: October 27th 2014 To:December 22nd 2014

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

suspension

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: weekly


- applied as a suspension

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in corn oil was demonstrated over a period of 7 days at room temperature.

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.:

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

Dose / conc.:

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

Dose / conc.:

1 000 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 with 300 and 1000 mg/kg bw (project no. 1OC0416/13C103), no effects up to 1000 mg/kg bw other than dose-dependent increase in copper content in livers. Adverse findings on liver histopathology in one of 8 animals at 1000 mg/kg bw could not be clearly attributed to treatment and were not reason enough to lower the highest dose for the main study. Parameters investigated were body weight, food consumption, clinical chemistry parameters, haematology and liver histopathology. The study has been entered in the section on toxicokinetics (IUCLID 7.1.1).
- Rationale for animal assignment (if not random): Randomization

Positive control:

no

Observations and examinations performed and frequency:

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

Sacrifice and pathology:

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: 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

Other examinations:

Staining for copper of liver histopathology samples.

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation post dosing

Mortality:

mortality observed, treatment-related

Description (incidence):

salivation post dosing

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males of test group 3 (1000 mg/kg bw/d) total white blood cell (WBC) counts and absolute lymphocyte counts were increased.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males: ALT,total bile acids, cholesterol increased

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

The content of the digestive tract showed a green discoloration. This finding was regarded to be caused by the test substance, a greenishyellow pigment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

liver, see figure

Histopathological findings: neoplastic:

no effects observed

Details on results:

The concentrations in corn oil were found to be in the range of 97 to 107% of the nominal concentration. Analyses demonstrated a recovery of 106% in the low dose (100 mg/kg bw/d), 105% in the mid dose (300 mg/kg bw/d) and 98% in the high dose (1000 mg/kg bw/d). Homogeneity of the test substance preparations was also demonstrated.

No parental animal died or was sacrificed moribund during the study period.

Green discolored feces were seen in all animals of the test groups 2 and 3 (300 and 1000 mg/kg bw/d). The sign came up first on study day 1 in test group 3 (1000 mg/kg bw/d) and study day 8 in test group 2 (300 mg/kg bw/d).
This green discoloration was regarded to be caused by the test substance, a greenishyellow pigment. It was regarded to be test substance related but not adverse. Furthermore, slight to severe salivation was observed during pre-mating in 8 males and 6 females in test group 3 (1000 mg/kg bw/d). Likewise during mating in 5 males in test group 2 (300 mg/kg bw/d), 9 males and 8 females in test group 3 (1000 mg/kg bw/d).
During post-mating slight to severe salivation was observed in 6 males in test group 2 (300 mg/kg bw/d) and 9 males in test group 3 (1000 mg/kg bw/d). The findings were considered to be related to treatment, most likely caused by bad taste of the test compound, and therefore assessed as being not adverse.

No adverse effects were noted in the functional observation battery.

For males of test group 3 (1000 mg/kg bw/d) total white blood cell (WBC) counts and absolute lymphocyte counts were increased.

At the end of the administration period in males of test groups 2 and 3 (300 and 1000 mg/kg bw/d) alanine aminotransferase (ALT) activities, total bile acid (TBA) and cholesterol levels were increased (ALT in test group 3 not statistically significantly increased). Cholesterol levels were already higher in males of test group 1 (100 mg/kg bw/d) although not statistically significantly. However, in this test group this was the only alteration and therefore it was regarded as treatment-related but not adverse (ECETOC Technical Report No 85, 2002). Additionally, in males of test group 3 (1000 mg/kg bw/d) total bilirubin levels were increased.

In the livers of test group 3 males and females (1000 mg/kg bw/day) increased numbers of single cell necrosis (normal or enlarged hepatocellular size, light eosinophilic, granular cytoplasm, often no nucleus detectable) or apoptosis was observed. This was also observed in one male animal of test group 2 (300 mg/kg bw/day). Furthermore, there was an increase in Kupffer cells, which occasionally formed aggregates. The Kupffer cells often
were of larger size compared to control animals. These findings were mainly observed in the left lateral lobe.
With the rubeanic acid stain greenish to black particles, most probably copper, located within the cytoplasm of hepatocytes of the treated animals but not the control animals could be detected.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Table 1: Total white and differential blood cell count in males at the end of the in-life phase

		0 mg/kg bw/d	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
WBC	Mean	4.50 v	4.76	4.61	6.44 *
[giga/L]	S.d.	0.77	0.56	0.81	1.36
day 42	N	5	5	5	5
	Median	4.55	4.54	4.52	5.74
NEUTA	Mean	0.73 k	1.10	1.07	1.16
[giga/L]	S.d.	0.22	0.53	0.24	0.22
day 42	N	5	5	5	5
	Median	0.63	1.00	0.98	1.15
LYMPHA	Mean	3.58 v	3.45	3.33	5.02 *
[giga/L]	S.d.	0.58	0.33	0.64	1.27
day 42	N	5	5	5	5
	Median	3.51	3.41	3.52	4.50
MONOA	Mean	0.08 k	0.10	0.11	0.12
[giga/L]	S.d.	0.04	0.02	0.05	0.02
day 42	N	5	5	5	5
	Median	0.09	0.11	0.10	0.12
EOSA	Mean	0.08 k	0.08	0.08	0.10
[giga/L]	S.d.	0.02	0.03	0.03	0.03
day 42	N	5	5	5	5
	Median	0.08	0.08	0.07	0.11
BASOA	Mean	0.01 k	0.01	0.01	0.01
[giga/L]	S.d.	0.00	0.01	0.00	0.01
day 42	N	5	5	5	5
	Median	0.01	0.01	0.01	0.01
LUCA	Mean	0.02 v	0.02	0.01	0.03
[giga/L]	S.d.	0.01	0.01	0.01	0.01
day 42	N	5	5	5	5
	Median	0.02	0.02	0.01	0.03
NEUT	Mean	16.1 k	22.5	23.4	18.4
[%]	S.d.	3.2	8.4	4.3	4.2
day 42	N	5	5	5	5
	Median	15.4	23.2	23.2	17.4
LYMPH	Mean	79.8 k	73.2	72.1	77.5
[%]	S.d.	4.1	8.3	4.1	4.2
day 42	N	5	5	5	5
	Median	79.9	72.5	72.7	78.4
MONO	Mean	1.8 k	2.1	2.2	1.9
[%]	S.d.	0.8	0.5	0.7	0.4
day 42	N	5	5	5	5
	Median	1.7	2.1	2.1	1.8
EOS	Mean	1.7 k	1.7	1.8	1.6
[%]	S.d.	0.4	0.4	0.5	0.5
day 42	N	5	5	5	5
	Median	1.6	1.8	1.9	1.5
BASO	Mean	0.2 k	0.1	0.2	0.2
[%]	S.d.	0.1	0.1	0.0	0.0
day 42	N	5	5	5	5
	Median	0.2	0.1	0.2	0.2
LUC	Mean	0.4 k	0.4	0.2	0.4
[%]	S.d.	0.2	0.1	0.1	0.1
day 42	N	5	5	5	5
	Median	0.3	0.4	0.2	0.3

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

v=KRUSKAL-WALLIS-WILCOX; k=KRUSKAL-WALLIS

Table 2: Enzymes in males at the end of the in-life phase

		0 mg/kg bw/d	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
ALT	Mean	0.64 v	0.61	1.13 **	1.47
[μkat/L]	S.d.	0.09	0.06	0.37	0.91
day 42	N	5	5	5	5
	Median	0.61	0.58	1.02	1.08
AST	Mean	1.50 k	1.48	2.03	2.67
[μkat/L]	S.d.	0.21	0.31	0.61	1.38

day 42	N	5	5	5	5
	Median	1.48	1.40	1.90	1.87
ALP	Mean	1.40 k	1.38	1.78	1.44
[μkat/L]	S.d.	0.30	0.27	0.53	0.48
day 42	N	5	5	5	5
	Median	1.27	1.41	1.89	1.62
GGT_C	Mean	7 k	7	8	8
[nkat/L]	S.d.	3	3	4	5
day 42	N	5	5	5	5
	Median	8	9	9	7

Conclusions:

The substance shows adverse effects on rat liver at high doses. These effects appear to be related to accumulation of copper.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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 (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[1-[[(2-hydroxyphenyl)imino]methyl]-2-naphtholato(2-)-N,O,O']copper

EC Number:

239-763-1

EC Name:

[1-[[(2-hydroxyphenyl)imino]methyl]-2-naphtholato(2-)-N,O,O']copper

Cas Number:

15680-42-9

Molecular formula:

C17H11CuNO2

IUPAC Name:

[1-[[(2-hydroxyphenyl)imino]methyl]-2-naphtholato(2-)-N,O,O']copper

Test material form:

solid: nanoform

Details on test material:

- Substance type: pigment
- Physical state: Solid / greenish yellow
- Name of test material (as cited in study report): Irgazin Yellow L 0800
- Lot/batch No.: 0005629903
- Analytical purity: 99.0 %
- Purity test date: 2013
- Expiration date of the lot/batch: unlimited stability under storage conditions
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Date of manufacture: 01 Dec 2011

Test animals

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
- Weight at study initiation: 220 g (females) and 310g (males)
- 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: October 27th 2014 To:December 22nd 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: weekly


- applied as a suspension (4mL/kg bw

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:

The stability of the test substance in corn oil was demonstrated over a period of 7 days at room temperature.

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: 14-15 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 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 (project no. 1OC0416/13C103), no effects up to 1000 mg/kg bw
- Rationale for animal assignment (if not random): Randomization

Positive control:

no

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation post dosing

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

liver

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

The male mating index was 100% in all test groups. Fertility was proven for all of the F0 parental males within the scheduled mating interval to
produce F1 litter. One male of test group 1 (No. 16 mated with female No. 116) and one male in test group 2 (No. 23 mated with female No. 123) did not generate implants in utero. The male fertility index was 100% in test groups 0 and 3 and 90% in test groups 1 and 2.

The female mating index calculated after the mating period for F1 litter was 100% in all
test groups.
The mean duration until sperm was detected (GD 0) was 1.7, 2.7, 2.5, 2.1 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 got pregnant with one exception in test group 1 and one exception in test group 2. Female animal No. 116 (test group 1) which was mated with male No. 16 and female No. 123 which was mated with male No. 23 did get sperm in vaginal smear but showed no implants. The female fertility index was 100% in test groups 0 and 3 and 90% in test groups 1 and 2.
The mean duration of gestation was similar in all test groups (22.2 days in test group 0; 22.1 days in test groups 1 and 2, 22.3 days 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.

The gestation index was 100% in test group 0, 1 and 3 and 89% in test group 2 (300 mg/kg bw/d). The latter represents 8 females with live pups on the day of birth out of 9 females pregnant. Also in the historical control data not always 100% of the pregnant females had live pups: the historical control data ranges from 89% to 100%..

Effect levels (P0)

1

Dose descriptor:

NOEL

Remarks:

fertility

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on fertility at the limit dose

2

Dose descriptor:

NOEL

Remarks:

general toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

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

Histopathological findings:

not examined

Details on results (F1)

The rate live birth indices were 100% in test group 0, test group 1 (100 mg/kg bw/d) and test group 2 (300 mg/kg bw/d) and 99% in test group 3 (1000 mg/kg bw/d). One pup of test group 3 was found stillborn. The values of test group 1, 2 and 3 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.

The postimplantation loss was 16.5% in test group 0, 4.8% in test group 1 (100 mg/kg bw/d), 12.7% in test group 2 (300 mg/kg bw/d) and 7.3% in test group 3 (1000 mg/kg bw/d).
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 (0.7% - 18.3%) of the historical control data.

The viability index indicating pup mortality during lactation (PND 0 - 4) was 96.7% (test group 0), 100% (test group 1, 100 mg/kg bw/d), 98.4% (test group 2) and 95.6% (test group 3). The indices were based on two pups of test group 3 (1000 mg/kg bw/d) and one pup in test group 1 (100 mg/kg bw/d) cannibalized and two pups (test group 2) and three pups (test group 3) found dead. 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.

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show significant differences between the control and the test substance-treated groups.

In test group 1 (100 mg/kg bw/d) one and test group 3 (1000 mg/kg bw/d) five female pups of one dam (animal no. 131) showed a discolored thymus. The color was described as white instead of gray. In test group 2 (300 mg/kg bw/d) one female pup showed a discolored liver (pale) and its thorax was fluid filled (blood coagulum). These findings were assessed as being signs occurred post mortem or spontaneous in nature and thereby not related to treatment.

Effect levels (F1)

Dose descriptor:

NOEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Parameters up to postnatal day 4 (screening study)

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The substance caused no adverse effects on fertility and offspring development in a screening study (OECD 422) in rats with gavage application of 100, 300 and 1000 mg/kg bw.