Reaction mass of m-cresol and 2-chloro-m-cresol and 6-chloro-m-cresol

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP compliant OECD guideline 416 study, tested with the source substance CAS 59-50-7. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chlorocresol

EC Number:

200-431-6

EC Name:

Chlorocresol

Cas Number:

59-50-7

Molecular formula:

C7H7ClO

IUPAC Name:

4-chloro-3-methylphenol

Details on test material:

- Name of test material (as cited in study report): 4-Chloro-3-methylphenol
- Physical state: Pastilles
- Analytical purity: 100%
- Lot/batch No.: CHT0126 and CHA0152
- Stability under test conditions: until 2006-10-07 and 2007-12-12, respectively.
Stability and homogeneity in diet approved for up to 15 days in 15 kg mixtures

Test animals

Species:

rat

Strain:

other: Wistar Crl: (WI) WU BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, Sulzfeld, Germany
- Age at study initiation: (P): 5-6 weeks, (F1): 4 weeks
- Weight at study initiation: (P) Males:113-149 g; Females: 95-122 g; (F1) Males: 61-120 g; Females: 62-110 g

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on mating procedure:

The first F0 or F1 male was caged overnight with the first F0 or F1 female from the same test group (and so on) for maximum 12 times.
Mating was performed up to 3 weeks or until spermatozoa were observed in vaginal smears taken the next morning. On day 4 of lactation litters were culled to 8 pups/litter. F1 offspring were nursed up to an age of four weeks. Some of them were selected for further treatment (12 weeks) and for breeding an F2a generation. A second mating of F1 animals was performed giving rise to F2b litters to clarify relatively low viability indices at 0 and 3000 ppm.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

F0 animals: from beginning of the study until sacrifice. F0 animals were treated beginning 10 weeks before mating.
F1 animals: from weaning until sacrifice
F2 animals: sacrifice after weaning
Parental F0 animals received the test substance for a period of about 10 weeks before mating and were then allowed to mate over a period of up to three weeks. F1 offspring were nursed up to an age of four weeks. Some of them were selected for further treatment (12 weeks) and for breeding a F2 generation.

Frequency of treatment:

daily (free access to food containing the test stubstance)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
750, 3000, 12000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
63.8, 247.8, 1043.0 mg/kg bw/d (F0 males pre-mating period)
Basis:
other: mean dose as calculated from the reported body weight and standard food intake values

Doses / concentrations 3

Remarks:

Doses / Concentrations:
74.5, 288.4, 1204.9 mg/kg bw/d (F1 males pre-mating period)
Basis:
other: mean dose as calculated from the reported body weight and standard food intake values

Doses / concentrations 4

Remarks:

Doses / Concentrations:
80.1, 298.2, 1189.7 mg/kg bw/d (F0 females pre-mating period)
Basis:
other: mean dose as calculated from the reported body weight and standard food intake values

Doses / concentrations 5

Remarks:

Doses / Concentrations:
90.4, 364.5, 1263.4 mg/kg bw/d (F1 females pre-mating period)
Basis:
other: mean dose as calculated from the reported body weight and standard food intake values

No. of animals per sex per dose:

25 per sex per dose (P and F1)

Control animals:

yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes, once daily for clinical signs and twice daily for mortality and morbidity.

BODY WEIGHT: Yes, Body weights were recorded directly prior to the first administration and thereafter weekly up to necropsy (males and females not pregnant) and during the pregnancy and lactation periods as follows:
- During pregnancy on Day p.c. 0, 7, 14 and 20.
- During lactation on Day p.p .0, 4, 7, 14, 21 and 28.
- On the day of scheduled necropsy.

FOOD CONSUMPTION: Yes, Males: Weekly from week 1 up to necropsy (except during mating period).
-Females: Weekly from week 1 up to mating.
During pregnancy Day p.c. 0-7; 7-14; 14-20.
During lactation Day p.p. 0-4; 4-7.

Oestrous cyclicity (parental animals):

Oestrus cycle length determination was done by evaluation of vaginal smears received daily over 19 consecutive days prior to the mating period. The smears were examined microscopically for large set-rated cells indicating that oestrus had occurred. This data was used to determine the oestrus cycle length and whether females were cycling properly.

Sperm parameters (parental animals):

Spermatological investigations were performed in all surviving F0 and F1 males of the 0 and 12000 ppm group on the day of necropsy.
The following spermatozoa parameters were assessed:
motility and viability, morphology, epididymal spermatozoa count, count of homogenisation-resistant spermatid heads in the testis

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, litters were culled to 8 pups/litter

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Number of live and dead pups, pup weight, external alterations, ano-genital distance (in F2a pups), sex of each pup, developmental milestones (balano-preputial separation, vaginal opening)

Postmortem examinations (parental animals):

GROSS NECROPSY
Necropsies were done on all rats. Implantation sites in F0 and F1 females were recorded.

ORGAN WEIGHTS: Selected organs were weighed in adult rats and weanlings: Adrenals, brain, epididymides, kidneys, liver, ovaries and oviducts, pituitary, prostate, seminal vesicle and coagulating glands, spleen, testes, thyroid/parathyroids, uterus w/ cervix

HISTOPATHOLOGY
The following organs and tissues were examined at least in the control and high-dose group (including ovarian follicle staging (only F1)). This included also all F0/F1 rats which died intercurrently and those which were sacrificed moribund.
Abnormalities, adrenals, brain, epididymides, oesophagus, kidneys, larynx, liver, ovaries and oviducts, pituitary, prostate, seminal vesicle and coagulating glands, skin in mammary region, testes, thyroid/parathyroids, trachea, uterus w/ cervix, vagina, head w/ scull cap

Postmortem examinations (offspring):

GROSS NECROPSY
Apparently abnormal tissues, if any, were fixed in all pups/weanlings. In F1, F2a and F2b weanlings, brain, spleen, thymus, uterus and kidneys of one male and one female out of the first necropsied 5 litters per group were preserved in formalin.

ORGAN WEIGHT
The brain, spleen, thymus and uterus of one male and one female per F2a and F2b litter were trimmed and weighed as soon as possible after dissection. The ratio of organ weights to body weights was calculated. Therefore, all these weanlings were weighed at day of necropsy.

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

Description (incidence and severity):

starting at 3000 ppm in F1 animals

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

starting at 3000 ppm in F1 animals

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

starting at 3000 ppm in F1 animals

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: At 12000 ppm F1 females ingested less than control animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

at 12000 ppm in F0 and F1 animals

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

Following effects were seen in animals of the different dose groups:
F0 animals: No effects were seen in F0 animals and F1 pups of control, low and mid dose.
At the high dose, findings in F0 males comprised of:
Reduced body weight during pre-mating; reduced glycogen stores and altered fat deposition in the liver and hyaline casts and tubulus dilation in the kidneys.
Findings in F0 females at the high dose level comprised of:
Reduced body weight during pre-mating, gestation and lactation; increased relative liver weights, reduced glycogen stores, hypertrophy and altered fat deposition in the liver; increased tubulus epithelium inclusion, tubulus dilation and basophilic tubules in the kidney; smaller ovaries, ovary athrophy/changed cycle and epithelium athrophy of the vagina.

F1 animals: No effects were seen in animals of control and low dose.
Findings in F1 males comprised of:
At the mid dose, reduced absolute and relative liver weights.
At the high dose, reduced body weight during pre-mating, reduced absolute and relative liver weights, reduced glycogen stores and altered fat deposition in the liver, increased weights of the seminal vesicles and necrosis, simple tubulus dilation and hyaline casts in the kidney.
The increased weights of the seminal vesicles noted in F1 males were not considered to be treatment related as examinations on sperms revealed no effect. The liver changes (reduced hepatocellular glycogen storage, reduced periportal fat storage, adaptive periportal hypertrophy) might reflect secondary catabolic effects in course of a significant body weight loss in high dose animals and are associated with decreased liver weights in 3000 and 12,000 ppm F1 males

Findings in F1 females comprised of:
At the mid dose, reduced body weight during lactation, hypertrophy and altered fat deposition of the liver and simple tubulus dilation and tubulus dilation of the kidney.
At the high dose, reduced body weight during pre-mating, gestation and lactation; dilated stomach/caecum and emaciation; reduced glycogen stores and altered fat deposition of the liver; necrosis, tubulus dilation and increased relative weights of the kidney, the adrenals and the spleen; reduced weight of ovaries, ovary athrophy/changed cycle (increased metoestrus, decreased dioestrus) reduced growing follicles and reduced corpora lutea
The statistically significantly increased relative weights of the adrenals and spleen in F1 females were not considered to be adverse as histological correlates are missing. The ovarian atrophy, increased metoestrus, decreased dioestrus and atrophy of the vaginal epithelium as well as the decreased number of growing follicles and corpora lutea in F1 rats are considered to be possibly secondary due to weight loss and are correlated with reduced ovary weights and/or smaller ovaries.

Effect levels (P0)

1

Dose descriptor:

LOAEL

Effect level:

1 043 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Basis for effect level: body weight; some organ weights Remark: 1043 mg/kg bw corresponds to 12000 ppm (mean dose value as calculated from the body weight and food intake values)

2

Dose descriptor:

LOAEL

Effect level:

1 190 mg/kg bw/day

Based on:

test mat.

Remarks:

(mean dose value as calculated from the reported body weight and food intake values)

Sex:

female

Basis for effect level:

other: Basis for effect level: body weight; some organ weights; smaller ovaries Remark 1190mg/kg bw/day corresponds to 12000 ppm (mean dose value as calculated from the body weight and food intake values)

3

Dose descriptor:

NOAEL

Effect level:

248 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Basis for effect level: No effects were seen at this dose level. Remark: 248 mg/kg bw/day corresponds to 3000ppm (mean dose value as calculated from the body weight and food intake values)

4

Dose descriptor:

NOAEL

Effect level:

288 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Basis for effect level: No effects were seen at this dose level. Remark: 288 mg/kg bw/day corresponds to 3000 ppm (mean dose value as calculated from the body weight and food intake values)

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

at 12000 ppm in F2a pups

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

starting at 3000 ppm in F2b pups

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

at 12000 ppm in F1 and F2a/F2b pups

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Details on results (F1)

Following effects were seen in animals of the different dose groups:
F1 pups: No effects were seen in F0 animals and F1 pups of control, low and mid dose
F1 pups: At the high dose reduced pup weights, reduced litter weights, reduced absolute and/or relative spleen and thymus weights were recorded.
The occurrence of developmental milestones (balano-preputial separation and vaginal opening) was delayed in F1 rats of the high dose group.

F2a and F2b pups: No effects were seen in animals of control and low dose.
Findings of the F2a pups comprised of:
At the high dose reduced pup weights, reduced absolute and/or relative spleen and thymus weights and clinical signs of toxicity. No effects were seen in pups of the lower dose ranges. The amount of autolytic pups for F2a pups was at the highest dose group higher than in the other groups.
Findings of the F2b pups comprised of:
At the high dose reduced pup weights, reduced litter weights, reduced absolute and /or relative spleen and thymus weights and clinical signs of toxicity. No effects were seen in pups of the lower dose ranges. At the mid dose reduced pup weights were noticed.

Effect levels (F1)

1

Dose descriptor:

LOAEL

Remarks:

pups (LOAEL for pup effect is based on the lowest maternal dose during gestation/lactation )

Generation:

F1

Effect level:

862 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Basis for effect level: pup/litter weight; spleen and thymus weights Remark: 862 mg/kg bw/day corresponds to 12000 ppm (mean dose value as calculated from the body weight and food intake values)

2

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

248 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Basis for effect level: some organ weights Remark: 248mg/kg bw/day corresponds to 3000 ppm (mean dose value as calculated from the body weight and food intake values)

3

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

298 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Basis for effect level: body weight; gross pathology and histopathology of liver and kidney Remark: 298 mg/kg bw/day corresponds to 3000 ppm (mean dose value as calculated from the body weight and food intake values)

4

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Basis for effect level: No effects were seen at this dose level. Remark: 75 mg/kg bw/day corresponds to 750 ppm (mean dose value as calculated from the body weight and food intake values)

5

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

90 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Basis for effect level: No effects were seen at this dose level. Remark: 90 mg/kg bw/day corresponds to 750 ppm (mean dose value as calculated from the body weight and food intake values)

6

Dose descriptor:

NOAEL

Remarks:

pups (NOAEL for pup effect is based on the lowest maternal dose during gestation/lactation)

Generation:

F1

Effect level:

54 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Basis for effect level: No effect was seen at this dose level. Remark: 54 mg/kg bw/day corresponds to 750 ppm (mean dose value as calculated from the body weight and food intake values)

Results: F2 generation

Effect levels (F2)

1

Dose descriptor:

LOAEL

Remarks:

pups (LOAEL for pup effect is based on the lowest maternal dose during gestation/lactation)

Generation:

F2a

Effect level:

982 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Basis for effect level:body weight; spleen and thymus weight Remark: 982 mg/kg bw/day corresponds to 12000 ppm (mean dose value as calculated from the body weight and food intake values)

2

Dose descriptor:

LOAEL

Remarks:

pups (LOAEL for pup effect is based on the lowest maternal dose during gestation/lactation)

Generation:

F2b

Effect level:

195 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Basis for effect level: body weight Remark 195 mg/kg bw/day corresponds to 3000 ppm (mean dose value as calculated from the body weight and food intake values)

3

Dose descriptor:

NOAEL

Remarks:

pups (NOAEL for pup effect is based on the lowest maternal dose during gestation/lactation)

Generation:

F2a

Effect level:

235 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Basis for effect level: No effects were seen at this dose level. Remark: 235 mg/kg bw/day corresponds to 3000 ppm (mean dose value as calculated from the body weight and food intake values)

4

Dose descriptor:

NOAEL

Remarks:

pups (NOAEL for pup effect is based on the lowest maternal dose during gestation/lactation)

Generation:

F2b

Effect level:

47 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Basis for effect level: No effect was seen at this dose level. Remark: 47 mg/kg bw/day corresponds to 750 ppm (mean dose value as calculated from the body weight and food intake values)

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of effects seen within the two generation study

	F0 rearing F1			F1 rearing F2a			F1 rearing F2b
Dose [ppm]	750	3000	12000	750	3000	12000	750	3000	12000
Parental animals
Body weight: Premating			↓			↓	-	-	-
Body weight: Gestation			↓			↓			↓
Body weight: Lactation			↓		↓	↓		↓	↓
Food intake: Premating						↓ F	-	-	-
Developmental milestones	-	-	-			→	-	-	-
Necropsy
Liver weights (absolute and relative)								↓ M	↓ M
Liver weights (relative)			↑ F						↑ F
Seminal vesicles weight (absolute and relative)									↑
Ovaries weight (absolute and relative)			↓	-	-	-			↓
Ovaries diminished in size			yes	-	-	-
Ovaries growing follicles	-	-	-						↓
Ovaries corpora lutea	-	-	-						↓
Stomach/cecum dilated									yes F
Emaciation									yes F
Ovary athropie/changed cycle			yes F						yes F
Vagina epithelium athrophy			yes F						yes F
Kidney, adrenals, spleen weight (relative)									↑ F
Liver hypertrophy			yes F					yes F	yes F
Liver reduced glycogen			yes						yes
Liver fat deposition changed			yes					yes F	yes
Kidney necrosis									yes
Kidney simple tubulus dilation								yes F	yes
Kidney tubulus epithel inclusion			↑ F						↑ F
Tubulus dilation			yes F					yes F	yes F
Kidney basophilic tubules			yes F						yes M
Hyaline casts/ tubulus dilation			yes M						↓ M
Litter/Pup data
Pup weights			↓			↓		↓	↓
Litter weights			↓						↓
Clinical signs						yes
spleen weights (absolute and/or relative)			↓			↓			↓
Thymus weights (absolute and/or relative)			↓			↓			↓
↓ = decrease; ↑ = increase; → = delayed - = not measured, yes = finding present F = females only, M = males only

The parameters of the reproductive performance such as insemination, fertility, gestation and rearing indices as well as gestation length were not influenced by the treatment with the test substance up to 12000 ppm.

There was no test substance-related reduction in viability and lactation indices up to 12000 ppm.

Table 2: Test substance intake by dams during gestation and lactation

Dietary concentration [ppm]	Generation (females)	Mean test substance consumption [mg/kg bw/day]
		day 14 to 20 p.c.	Day 0 to 4 p.p.
750	F0	54.2 ± 3.08	89.2 ± 32.97
	F1 rearing litter F2a	55.8 ± 7.55	83.0 ± 31.65
	F1 rearing litter F2b	46.6 ± 6.78	86.7 ±27.61
3000	F0	216.8 ± 14.60	320.7 ± 110.53
	F1 rearing litter F2a	235.3 ± 35.55	345.1 ± 125.47
	F1 rearing litter F2b	195.1 ± 24.36	279.7 ± 84.01
12000	F0	861.8 ± 85.46	1385.8 ± 431.38
	F1 rearing litter F2a	982.3 ± 199.80	1582.8 ± 525.45
	F1 rearing litter F2b	773.4 ± 100.17	1495.3 ± 417.4