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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:
key study
Study period:
03-09-2012 to 30-11-2012
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: para-Menthane
Chemical name Cyclohexane,1-methyl-4-(1-methylethyl)/1-isopropyl-4-methylcyclohexane
Appearance: colourless liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar Han (IGS)
- Source: Charles River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 11-12 wks
- Weight at study initiation: Males: 296-335 g; Females: 197-223 g
- Housing:
three male animals in open macrolon cages type 2000P, TechniPlast (size slightly larger than GV-SOLAS Type IV)
females single housed with their litters in open macrolon cages type III.
- Diet : No. 1314 TPF (Altromin Spezialfutter GmbH & Co. KG, 32791 Lage), ad lib.
- Water: Sterilised community tap water, ad lib.
- Acclimation period: 5-9 days
- Pregnancy status females: Nulliparous, non-pregnant

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10/h
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 03-09-2012 to 30-11-2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): as solubility in water limited, corn oil was selected
- Concentration in vehicle: 30.5 g/L, 91.2 g/L and 273.3 g/L
- Application volume: 4 mL/kg bw

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until proof of pregnancy or 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility for the high dose group.

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Stability of standard concentrations over a period of 7 days was investigated. All concentrations were within 96-102% of nominal.
Method GC/FID on ZB624 column. Two isomers were identified and the summation of both peaks was used for quantification
Duration of treatment / exposure:
Exposure duration:
Males: 14 days prior to mating; upto 14 days during mating and 14 days post-mating
Females: 14 days prior to mating; upto 14 days during mating; average 21 days of gestation; 4 days of lactation
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 13-14 weeks first mating (15-16 weeks second mating of high dose group)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 111, 333 and 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a dose range finding study with increasing doses (upto 2000 mg/kg bw) over the 18 day study period. The animals showed toxic effects only at the highest dose level.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (IRWIN test) in a standard arena
Appearance (general status, physiology, autonomic functions, neurology, tonus, E1-E4, Motoric / exploration behaviour (M1-M2), Excitation (R) andAbnormal behaviour (A1-A4)

BODY WEIGHT: Yes
- Time schedule: weekly

FOOD CONSUMPTION: yes
Time schedule: weekly

WATER CONSUMPTION: Yes
Time schedule: weekly

HAEMATOLOGY: yes on 5 males and 5 females at end of premating period
Leukocytes, Erythrocytes, Haemoglobin , Haematocrit ,Mean corpuscular volume (MCV) , Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin conc. (MCHC), Thrombocytes, Reticulocytes, Neutrophil granulocytes, Lymphocytes, Monocytes, Eosinophils. Basophils

CLINICAL CHEMISTRY: yes on 5 males and 5 females at end of premating period
Alkaline phosphatase, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Cholesterol, Urea, Sodium, Potassium, Calcium, Chlorid, Glucose, Total protein, Albumin, Globulin, A/G ratio, Creatinine, Bile acids

OTHER:
Behavioural tests
Grip strength and beam walking: last exposure week on 5 males and 5 females

Reproduction parameters:
Females showing evidence of copulation, Females achieving pregnancy , no. of conceiving days, no. of days of pregnancy, no. of dams with live young born/with live young at day 4 pp, Corpora lutea / dam, Implants/dam , Live pups/dam at birth/at day 4, Sex ratio (m/f) at birth / at day 4
Loss of offspring, Pre-implantation (corpora lutea minus implantations), pre-natal (implantations minus live births), Post-natal (live births minus alive at post natal day 4)
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, live births, postnatal mortality, weight at birth / at day 4, physical or behavioural abnormalities

Postmortem examinations (parental animals):
ORGAN WEIGHTS:
Brain, Heart, Thymus, Liver, Spleen, Kidney, Adrenals, Prostate, Epididymides, Testes, Ovaries

GROSS NECROPSY on all animals
Gross lesions, Oesophagus, Trachea, Thyroid, Stomach, Thymus, Liver, Spleen, Duodenum, Jejunum, Ileum (with Peyer’s patches), Cecum, Colon, Rectum, Lymph nodes(mesenteric), Kidney, Adrenals, Urinary bladder, sternum, spinal cord, whole brain, cerebrum, cerebellum, Peripheral nerve, Bonemarrow, Pons, Skeletal muscle, Heart, Lungs, Vagina, Epididymides, Prostate/uterus/cervix, Testes/ovary, eye

HISTOPATHOLOGY:
ovaries, testes and epididymides of 5 animals of the high dose group and the control group
all organs of 5 males and 5 females of the high dose group and the control group
Postmortem examinations (offspring):
not specified
Statistics:
Anova with Dunnett’s t-test

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
reduced pregnancy rate in high dose animals

Details on results (P0)

MORTALITY (PARENTAL ANIMALS):
1000 mg/kg bw :1 female on day 14 , 1 male on day 19, 1 female on day 4 of lactation (gavage error, KIE)
none in other dose groups and controls

CLINICAL SIGNS/BEHAVIOURAL TESTS:
no treatment related effects

BODY WEIGHT (PARENTAL ANIMALS):
Males: sign. decrease during pre-mating and post-mating period at 1000 mg/kg bw;
Females: no treatment related effects, reduced during gestation in pregnant females of 1st mating (n=3)

FOOD CONSUMPTION (PARENTAL ANIMALS):
Males: increased at 1000 mg/kg bw
Females: no treatment related effects

WATER CONSUMPTION (PARENTAL ANIMALS):
Males: dose related increase at 111, 333 and 1000 mg/kg bw
Females: increased at 1000 mg/kg bw until day 14 of gestation

CLINICAL CHEMISTRY
Increased ALAT in males and females and decreased ALP in females at 1000 mg/kg bw
Slightly increased total protein in females of all dose groups

HAEMATOLOGY
Decreased Hb in females at 333 and 1000 mg/kg bw (no effects on MCH and MCHC)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
see table (under off-spring)

Mean reproduction data females
0 mg/kg bw 111 mg/kg bw 333 mg/kg bw 1000 mg/kg bw
Number of dams examined 12/12 12/12 11/12 8/11
Corpora lutea/dam 14 14 14 12
Implantation sites/dam 10 10 11 5
Pre-implantation loss/dam 4 4 3 7
Post-implementation loss/
dam 1 0 1 0
Number of dams delivering
live pups 11 12 11 6

Increased pre-implantation loss in females at 1000 mg/kg bw

ORGAN WEIGHTS (PARENTAL ANIMALS)
Kidney: increased in males at 333 and 1000 mg/kg bw
Liver: dose related increase in males (sign at 333 and 1000 mg/kg bw)
Thymus: decreased in males at 333 and 1000 mg/kg bw
Ovaries/uterus: increase at 1000 mg/kg bw

GROSS PATHOLOGY (PARENTAL ANIMALS)
no treatment related findings (all findings were incidental and within normal background)

HISTOPATHOLOGY (PARENTAL ANIMALS)
kidneys: hyaline droplets and/or casts in all males at 111, 333 and 1000 mg/kg bw
other findings were considered within normal ranges

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
111 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: hyaline droplets and casts in kidney
Key result
Dose descriptor:
NOAEL
Effect level:
333 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: effects at the high dose level were limited to decreased body weight (gain) during gestation, increased water consumption and a slight increase of the weight of the uteri
Dose descriptor:
NOAEL
Effect level:
333 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The NOAEL for reproductive effects is set at 333 mg/kg bw, as at the highest dose the pregnancy rate is strongly decreased.
Remarks on result:
other: Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified

Any other information on results incl. tables

Dosage

0 mg/kg bw

111 mg/kg bw

333 mg/kg bw

1000 mg/kg bw

Pairs started

12

12

12

11

Females achieving pregnancy

11

12

11

6

SP detected days 1 – 5(2)

12

11

10

13

SP detected days 6 and more(1) (2)

0

1

1

2

Pregnancy ≤ 21 days

0

1

0

0

Pregnancy = 22 days

8

8

8

4

Pregnancy = 23 days

3

3

2

2

Dams with live young born

11

12

11

6

Dams with live young at day 4pp(3)

11

12

11

5

Implants/dam (mean)

9.9

9.8

10.8

5.0

Live pups/dam at birth (mean)

10.1

9.4

10.0

5.7

Live pups/dam at day 4 (mean)

10.0

9.4

9.9

6.4

Litter weight at birth (mean)

64.7

58.8

59.4

34.6

Litter weight at day 4 (mean)

110.7

102.5

100.4

64.7

Pup weight at birth (mean)

6.4

6.3

6.0

6.3

Pup weight at day 4 (mean)

11.3

11.2

10.1

10.0

Total of pups born day 0 (count)

111

113

110

34

Stillborn (count)

1

0

1

0

Pups alive day 4(4)

110

113

109

32

Sex ratio (M/F)

53/59

51/62

46/63

16/18

1last day of mating period

2differences in sum may occur in case an animal achieved pregnancy without sperm plug detected

3the offspring of one mother died because the mother was ill. Illness was not test item related

42 pups died in the HD group because the mother was moribund

Applicant's summary and conclusion

Conclusions:
The NOAEL based on repeated dose and reproduction toxicity is set at 333 mg/kg bw.
Executive summary:

Daily oral administration of the test substance to Wistar rats at dose levels of 111, 333, and 1000 mg/kg body weight over a period of 44 to 57 days resulted at the highest dose level in a reduction of the body weight gain and an increased water and food consumption in male animals. The water consumption was increased in females of the high dose group. Test item related changes in organ weight were found in the liver, the kidney and thymus of male animals. Furthermore, in male rats the histopathological examination indicated a nephrotoxic effect in all dose groups. At the highest dose level, the test item also showed a potential

effect on the metabolism of the liver as well as an influence on uterus weight of female rats.

The numbers of pregnancies and the litter size were also reduced in animals treated at the highest dose level. An increased pre-implantation loss was seen in high dosed females.

The NOAEL is 333 mg/kg bw for females. In males based on the effects on the kidney a LOAEL of 111 mg/kg bw was derived. As these effects are considered of less relevance for humans, the NOAEL used in reisk assessment is set at 333 mg/kg bw.