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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No study available.

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

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)

Remarks:

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

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

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

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

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

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.

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: 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

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.

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)

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)

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)

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)

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

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.

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)

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)

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)

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)

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)

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)

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)

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)

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)

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)

Reproductive effects observed:

not specified

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

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

The database for this endpoint is deemed to be acceptable and sufficient for non-classification.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The relevant data on toxicity to reproduction for Preventol KMX and the read across substance 4-chloro-3-methylphenol are summarized in the table below. The read across justification and additional data for m-cresol are attached to the Chemical Safety Report in Annex I. As m-cresol is only a minor component of Preventol KMX. Therefore these data are not considered for the assessment of the toxicity to reproduction of Preventol KMX.

Table 1: Comparison of relevant data on reproductive toxicity of Preventol KMX and 4-chloro-3-methylphenol

Endpoint	Preventol KMX	4-chloro-3-methylphenol
Toxicity to reproduction	Read across to 4-chloro-3-methylphenol	No effects on fertility observed, NOAEL 2-generation study: 268 mg/kg bw/day (P, male/female) 54 mg/kg bw/day (F1 pups, male/female), 83 mg/kg bw/day (F1, male/female), 235 mg/kg bw/day (F2a pups), 47 mg/kg bw/day (F2b pups)
Developmental toxicity	Read across to 4-chloro-3-methylphenol	NOAEL developmental toxicity oral, rat: 100 mg/kg bw/day

 

No data on toxicity to fertility for Preventol KMX (reaction mass of 6-chloro-m-cresol, 2-chloro-m-cresol and m-cresol) are available. The possibility of a read-across to Preventol CMK (4-chloro-3-methylphenol) in accordance with Regulation (EC) No 1907/2006 Annex XI 1.5. Grouping of substances and read-across approach was assessed. There it is given that a read-across approach is possible for substances, whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity. Preventol KMX contains of approximately 58% 6-chloro-m-cresol, 30% 2-chloro-m-cresol, 9% m-cresol and smaller amounts of p-cresol (approx. 2.5%), 4-chloro-3-methylphenol (approx. 0.5%) and other impurities (approx. 0.5%). A literature search and QSAR predictions were performed for 2-chloro-m-cresol, 6-chloro-m-cresol using m-cresol and 4-chloro-3-methylphenol (=4-chloro-m-cresol) as reference substances. Next to physico-chemical properties, environmental fate, ecotoxicity, toxicity, and metabolism of chlorocresols and chlorophenols in various databases, special emphasis was set on determining the effects of different positions of chlorine on aromatic ring structures as this is the determinant difference between the 3 chlorocresol constituents, which may influence outcome in physicochemical and toxicological behaviour. The aim was to evaluate the read-across from 4-chloro-3-methylphenol to Preventol KMX and to ensure a safe and legally valid analogue approach.

The chlorocresols are found to be similar in structure and the available data shows that the substances are similar in physico-chemical properties, environmental fate, metabolism and predicted affected pathways. Literature revealed no evidence of relevant influence of the position of chlorination on ring structures on the toxicological properties of a substance. Thus, the position of the chlorination is not regarded to significantly modulate the toxic effects of the chlorocresols. Since the discussed chlorocresols differ only in the position of the chlorination, the toxicological properties of the substances can be expected to be similar. Hence, 4-chloro-3-methylphenol is determined as a suitable read-across substance to predict toxicity endpoints in the chemical risk assessment of reaction mass of 6-chloro-m-cresol, 2-chloro-m-cresol and m-cresol (Preventol KMX). A detailed justification for the read-across is provided in the technical dossier (see IUCLID Section 13), as well as in the Chemical Safety Report (see Part B).

Available study of interest for 4-chloro-3-methylphenol:

The purpose of the GLP compliant two generation rat feeding study with doses of 750, 3000 and 12000 ppm 4-chloro-3-methylphenol was to evaluate possible effects of 4-chloro-3-methylphenol on the entire reproduction process in Wistar rats (Eiben, 2006). 4-chloro-3-methylphenol was administered to groups of 25 male and 25 female rats at concentrations of 750, 3000, or 12,000 ppm in their diet.

Parental F0 animals received the test substance for a period of about 10 weeks and were then allowed to mate over a period of up to three weeks. Females were further treated during gestation and weaning. 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 was done on F1 rats to clarify relatively low viability indices at 0 and 3000 ppm. Generally, the test substance was administered to the animals up to necropsy.

Mortality, clinical signs of toxicity, body weights food and water intake as well as reproduction parameters such as mating performance, fertility, gestation, rearing, oestrus cycling and sperm analyses were examined in F0 and F1 rats. Furthermore, litter parameters such as litter size, percentage of males born and pup weight at birth as well as viability and lactation indices, body weight gain and clinical signs were studied in F1, F2a and F2b offspring. Developmental milestones were evaluated in F1 post weanlings and ano-genital distance was measured in F2a pups. Necropsies were done on all rats. Implantation sites in F0 and F1 females were recorded. Selected organs were weighed of adult rats and weanlings and histopathology including ovarian follicle staging (only F1) was performed in a number of organs of F0 and F1 parental rats.

At the high dose group some findings in male and female animals were noticed.

No changes in clinical behavior and appearance of parental F0 rats were evident up to 12000 ppm.

At 12000 ppm increased water intake was seen in two F1 females during lactation. In F0 and F1 rats of the high dose group statistically significant decreased body weights were noted. At necropsy this led to some emaciated F1 females, which showed partly also a dilated stomach and/or caecum. A statistical significant decrease in body weight gain was noted during lactating in F1 females of the mid dose group from Day 0 p.p. to Day 4 p.p. In addition F1 females of the high dose ingested less diet as controls.

No adverse effects on body weights were evident in F0 rats up to 3000 ppm and F1 rats at 750 ppm.

In the liver, periportal cytoplasmic change, often coincided with reduced hepatocellular glycogen storage, was increasingly found in high dosed males of both generations. Simultaneously, periportal fat storage was reduced in favour of a more diffuse pattern. In contrast, adaptive periportal hypertrophy/eosinophilia occurred in F0 females of the high dose group and in F1 females increasingly at 3000 ppm and above. Increased relative liver weights occurred in females of the high dose group. Hepatocellular glycogen storage was reduced in both generations at 12,000 ppm. Changes in the fat storage became slightly evident in F1 females at 3000 ppm and above. These liver changes might also 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.

In the kidneys, papillary necroses were found in both genders of the F1 generation at 12,000 ppm. Simple dilation of the papillary tubules was increased at 12,000 ppm in both male generations and in the female F1 generation at 3000 ppm and above. These findings are interpreted as adverse. Brownish inclusions in the proximal tubules and dilated cortico-medullary tubules were only found in females and raised at 12,000 ppm in both generations, a small number of F1 females also showed dilated cortico-medullary tubules at 3000 ppm. Secondary changes belonging to chronic progressive nephropathy (CPN) either increased (F0 females: basophilic tubules) or decreased (F0/F1 males: hyaline casts/dilated tubules; F1 males: basophilic tubules) under high dose treatment.

In 12,000 ppm F1 females, relative kidney weights were increased. Investigation of animals without progeny did not demonstrate any compound-related aetiology.

At the high dose increased weights of the seminal vesicles were noted in F1 males. Examinations on sperms revealed no treatment-related effect. In F0 and F1 females of the high dose group ovarian atrophy, increased metoestrus, decreased dioestrus and atrophy of the vaginal epithelium appeared. Histopathological evaluations of ovarian follicles and corpora lutea revealed a statistically significant decrease in the number of growing follicles and corpora lutea in F1 rats. These findings are discussed as presumably secondary due to weight loss and are correlated with reduced ovary weights and/or smaller ovaries.

Although marked effects were noticed in animals of the high dose group 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 12,000 ppm. There was no test substance-related reduction in viability and lactation indices up to 12,000 ppm.

At the high dose group depressed pup and litter weights occurred in all generations. In addition, the occurrence of developmental milestones (balano-preputial separation and vaginal opening) was delayed in F1 rats of the high dose group. No effect was seen at measurements of the ano-genital distance in F2a pups.

The spleen and thymus weights were decreased in nearly all pup generations at 12,000 ppm. F2a pups of this dose group exhibited respiration sounds and blue discolorations. At 3000 ppm slightly reduced body weights were observed for female F2b pups. At 12,000 ppm more autolytic F2a pups were found than in the other groups.

The decrease in the number of growing follicles and corpora lutea in F1 females at the high dose group are thought to be correlated with the weight loss and to be a result of the resulting decreased ovary weights and/or smaller ovaries. In addition almost all pup related findings (decreased pup and litter weights, decreased spleen and thymus weights) were evident at the high dose group only, except for slightly reduced body weights of F2b pups of the mid dose where also marked maternal toxicity was observed. At dose levels of no or moderate maternal toxicity no effects in pups were evident.

Thus, classification of Preventol KMX according to the dangerous substance directive 67/548/EEC and according to CLP 1272/2008/EC is not required.

Short description of key information:
No data on toxicity to reproduction for Preventol KMX are available. However, a read-across approach to 4-chloro-3-methylphenol was judged to be scientifically reliable (for details see Discussion below).
A reliable, GLP compliant two generation rat feeding study with doses of 750, 3000 and 12000 ppm 4-chloro-3-methylphenol in the diet was performed according to OECD Guideline 416. No effects on fertility were evident. Thus, classification of Preventol KMX according to the dangerous substance directive 67/548/EEC and according to CLP 1272/2008/EC is not required.

Effects on developmental toxicity

Description of key information

No data on developmental toxicity for Preventol KMX are available. However, a read-across approach to 4-chloro-3-methylphenol was judged to be scientifically reliable (for details see Discussion below).
In a reliable, GLP compliant OECD guideline 414 study 4-chloro-3-methylphenol was applied to female  wistar rats at doses of 30, 100 and 300 mg/kg bw via gavage from day 6 to day 15 of gestation. On day twenty of gestation females delivered by caesarean section. The NOAEL for maternal toxicity was 100 mg/kg bw and the NOAEL for developmental toxicity was 100 mg/kg bw. 
Thus, classification of Preventol KMX according to the dangerous substance directive 67/548/EEC and according to CLP 1272/2008/EC is not required.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

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 414 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar (Bor:WISW(SPF Cpb))

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Borchen, Germany
- Age at study initiation: no data
- Weight at study initiation: 186 - 248 g

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous tylose solution

Details on exposure:

VEHICLE
- Concentration in vehicle: 3.0, 10.0, 30.0 mg/mL
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Virgin female rats were mated overnight individually with males. Females revealing vaginal plugs or spermatozoa in vaginal smears in the following morning were designated as being in day 0 of gestation.

Duration of treatment / exposure:

from day 6 to day 15 of gestation

Frequency of treatment:

daily

Duration of test:

20 days (On day 20 of gestation animals delivered by caesarean section)

Remarks:

Doses / Concentrations:
30, 100, 300 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

25

Control animals:

yes, concurrent no treatment

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
From day 0 to 20 of gestation: twice daily (once on weekends and bank holidays)

BODY WEIGHT: Yes
Day 0 p.c.; Days 6 to 15 p.c.: once daily; day 20 p.c.

FOOD CONSUMPTION: Yes
Days of gestation: 0-6, 6-11, 11-16 and 16-20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organ examined: Uterus weight

OTHER: Water consumption assessment was performed during inspections by visual inspection of the quantities left over.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Other: Individual weights and outward appearance of placentas

Fetal examinations:

Examination of the fetuses included:
Determination of sex, weight, occurrence of malformations discernable from the outside and other findings deviating from standard, occurrence of visceral malformations (investigations of half of the foetuses according to the modified WILSON technique, occurrence of changes in the abdominal and thoracic organs and skeletal system (evisceration and evaluation of the other foetuses according to the DAWSON technique)

Indices:

Sex ratio (m:f)

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: at 300 mg/kg bw/d

Details on maternal toxic effects:
No treatment related effects were seen in animals of the 0 and 30 mg/kg bw/d dose group.
CLINICAL SIGNS:
100 mg/kg bw/d: on days 8 and 16 two animals showed laboured breathing
300 mg/kg bw/d: from day 8 post coitum (p.c.). marked clinical signs (rough coat, sunken flanks, bloody muzzle, laboured breathing, reduced mobility, high-stepping gait) were noted. From day 6 p.c. the following signs occurred from about 10 minutes until 1 hour after application: lying on side, somnolence, abdominal position, spastic convulsion. In addition, one animal showed gasped breathing.
MORTALITY:
0 - 100 mg/kg bw/d: no mortalities occurred
300 mg/kg bw/d: Six animals died during the study period, one of them was sacrificed in moribund condition.
FOOD INTAKE:
100 mg/kg bw/d: statistically significant reduced food intake during the application period.
300 mg/kg bw/d: statistically significant reduced food intake during the application and gestation period.
WATER INTAKE:
100 mg/kg bw/d: reduced in one animal on day 9 p.c.
300 mg/kg bw/d: reduced for several animals from day 9 p.c., in some cases lasting for 1-6 days.
URINE/FAECES EXCRETION:
100 mg/kg bw/d: increased urine excretion in some isolated cases from day 9 p.c.; reduced amount of faeces over a period of 1-5 days in several animals from day 9 p.c.
300 mg/kg bw/d: increased urine excretion in several animals from day 8 p.c. for a period of 1-5 days; reduced amount of faeces in several animals from day 8 p.c. (lasting for 1-5 days).
BODY WEIGHT:
100, 300 mg/kg bw/d: body weight gain was reduced during the treatment period. Body weight development during pregnancy as well as corrected body weight gain was statistically significant reduced.
GROSS PATHOLOGY:
0 - 100 mg/kg bw/d: no effects
300 mg/kg bw/d: Of the 6 animals that died or were sacrificed moribund during the study period, 2 and 3 animals had inflated intestines and bloody vaginas, respectively. Additional significant findings were noted in these animals. No findings were observed in the surviving animals of this dose group. For further details see Table 1 under: "Any other information on results".

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: at 300 mg/kg bw/d

Details on embryotoxic / teratogenic effects:
EMBRYOTOXICITY:
0 - 100 mg/kg bw/d: No effects
300 mg/kg bw/d: Reduced foetal weight. Due to an increased rate of resorption, the gestation rate and number of foetuses were also reduced.
TERATOGENICITY: No effects.
For further details see Table 2 under: "Any other information on results".

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Overview of maternal effects in the prenatal developmental toxicity study

Parameter	Control data		30 mg/kg bw/d	100 mg/kg bw/d	300 mg/kg bw/d	Dose-response + / –
	historical	0 mg/kg bw/d
Number of dams examined	-	25	25	25	25
Clinical signs of toxicity
audible breathing sounds	-	0	0	1	4	+
gasping breathing	-	0	0	1	1	+
bloody lip	-	0	1	0	0	-
rough coat	3	0	0	0	7	+
bloody muzzle	1 (nose)	0	0	0	4	+
sunken flanks	1	0	0	0	3	+
reduced motility	-	0	0	0	2	+
abdominal knots	-	0	1	0	0	−
bloody forelimbs	-	0	0	0	1	−
high-stepping gait	-	0	0	0	1	+
reduced water intake	7#	0	0	1	12	+
light-brown, hard faeces	4	1	0	0	0
small amount of faeces	4	0	0	4	14	+
increased urine excretion	5#	0	0	3	9	+
after application
gasping breathing	-	0	0	0	1	+
lying on side	-	0	0	0	7	+
somnolence	-	0	0	0	13	+
abdominal position	-	0	0	0	9	+
spastic convulsion	-	0	0	0	5	+
Mortality of dams	-	0	0	0	6	+
Body weight gain [g] Mean day 0 – 20	73.0 - 101.9	98.5	95.8	90.9	66.8*	+
Body weight gain [g] corrected, day 0 – 20	-	37.6	35.3	31.0	13.7***	+
Mean food consumption (Day 0-20) [g/rat/day]	-	18.5	18.4	18.0	15.5***	+
Pregnancies	7-24	22	24	22	24	-
Necropsy findings in dams dead before end of test
reddened oesophagus	-	0	0	0	1	+
suppurative foci in lung tissue	-	0	0	0	1	+
fluid in thorax	-	0	0	0	1	+
thorax filled with serous fluid	-	0	0	0	1	+
stomach appears smaller	-	0	0	0	1	+
stomach + intestines extremely distended	-	0	0	0	1	+
reduced spleen size	-	0	0	0	1	+
gas-inflated intestines	-	0	0	0	2	+
bloody vagina	1	0	0	0	3	+
organs autolytic	-	0	0	0	1	+
Necropsy findings in dams at termination
Ovariary cysts	1	2	0	0	0	−
intestinal worms	56	3	8	7	6	-
# Skeletal retardations Statistically significant difference from controls: *p < 0.05; ** p < 0.005; *** p < 0.001

Table 2: Overview of developmental effects in the prenatal developmental toxicity study

Parameter	Controldata		30 mg/kg bw/d	100 mg/kg bw/d	300 mg/kg bw/d	Dose-response + / –
	Historical (1984-1990)	0 mg/kg bw/d
Corpora lutea[mean no./dam]	-	13.0	13.2	12.4	12.6
Implantations[mean no./dam]	8.3 - 12.5	11.4	11.3	11.2	10.6	–
Resorptions[mean no./dam]^a	0.3 - 2.3	0.6	0.8	0.6	1.8***	+
Resorptions[mean no./dam]^b		0.6	0.8	0.6	0.7	-
Foetuses[mean no./dam]	7.6 - 11.7	10.7	10.5	10.6	9.9	–
Foetus weight(mean) [g]^b	3.17 - 3.68	3.71	3.69	3.65	3.42**	+
Placenta weight[mean/dam] [g]^b	0.55 - 0.68	0.62	0.65	0.62	0.60	–
Skeletal changes [mean foetus no/dam]^b	1.44 - 3.18#	2.09	1.46	2.18	1.38	−
Malformations [mean foetus no/dam]^b	0.00 - 0.39	0.14	0.08	0.05	0.38	−
Sex ratio (m:f)^b	-	1:0.8	1:0.9	1:1.22	1:1.03	-
^awith implantations ^bwith live foetuses # Skeletal retardations Statistically significant difference from controls: *p < 0.05; ** p < 0.005; *** p < 0.001

Conclusions:

The test substance had no effect on intrauterine development at doses causing no maternal toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The database for this endpoint is deemed to be acceptable and sufficient for non-classification.

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

No data on toxicity to reproduction for Preventol KMX (reaction mass of 6-chloro-m-cresol, 2-chloro-m-cresol and m-cresol) are available. The possibility of a read-across to Preventol CMK (4-chloro-3-methylphenol) in accordance with Regulation (EC) No 1907/2006 Annex XI 1.5. Grouping of substances and read-across approach was assessed. There it is given that a read-across approach is possible for substances, whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity. A literature search and QSAR predictions were performed for 2-chloro-m-cresol, 6-chloro-m-cresol using m-cresol and 4-chloro-3-methylphenol (=4-chloro-m-cresol) as reference substances. Next to physico-chemical properties, environmental fate, ecotoxicity, toxicity, and metabolism of chlorocresols and chlorophenols in various databases, special emphasis was set on determining the effects of different positions of chlorine on aromatic ring structures as this is the determinant difference between the 3 chlorocresol constituents, which may influence outcome in physicochemical and toxicological behaviour. The aim was to evaluate a registration strategy based on the read-across from 4-chloro-3-methylphenol to Preventol KMX and to ensure a safe and legally valid analogue approach.

The chlorocresols are found to be similar in structure and the available data shows that the substances are similar in physico-chemical properties, environmental fate, metabolism and predicted affected pathways. Literature revealed no evidence of relevant influence of the position of chlorination on ring structures on the toxicological properties of a substance. Thus, the position of the chlorination is not regarded to significantly modulate the toxic effects of the chlorocresols. Since the discussed chlorocresols differ only in the position of the chlorination, the toxicological properties of the substances can be expected to be similar. Taking all this together, this validates the attempt to make a safe analogue approach from 4-chloro-3-methylphenol to 2-chloro-m-cresol and 6-chloro-m-cresol. Hence, 4-chloro-3-methylphenol is determined as a suitable read-across substance to predict toxicity endpoints in the chemical risk assessment of reaction mass of 6-chloro-m-cresol, 2-chloro-m-cresol and m-cresol (Preventol KMX). A detailed justification for the read-across is provided in the technical dossier (see IUCLID Section 13) as well as in the Chemical Safety Report (see Part B).

Available studies of interest for 4-chloro-3-methylphenol:

In a reliable, GLP compliant OECD Guideline 414 study 4-chloro-3-methylphenol was applied to female wistar rats at doses of 30, 100 and 300 mg/kg bw via gavage (10 mL/kg bw) from day 6 to day 15 of gestation (Bartmann, 1991).The test substance was applied in 0.5% aqueous tylose solution. On day twenty of gestation females delivered by caesarean section. Investigations were performed on general tolerance of the test substance by the dams and the examination of the uterus weight upon sacrifice. Fetal examinations comprised of determination of sex and weight, occurrence of malformations (discernible from the outside) and other findings deviating from standard and the occurrence of visceral malformations.

No effects were noted in animals of the low dose. At the mid dose females showed laboured breathing after application. At the high dose group females exhibited marked clinical signs (rough coat, sunken flanks, bloody muzzle, laboured breathing, reduced mobility, high-stepping gait). Within 1 hour after application, animals of this group showed additional clinical signs of more frequently lying on side, somnolence, abdominal position, spastic convulsions, gasping breathing. Six of 25 females died during the study period, one of them was sacrificed in moribund condition.Upon necropsy among other findings inflated intestines and bloody vaginas were seen in these animals. No findings were observed in the surviving animals of this dose group.

In the mid and the high dose groups the food and water intake and the excretion of faeces and body weight gain was diminished while the urine excretion was increased.

No effects on the weight and external appearance of placentas, sex ratio of foetuses and development of the skeletal system were seen in any dose group. Gestation and resorption rates, number and weight of foetuses as well as number and kind of malformations were not affected at and below 100 mg/kg bw/day. In animals of the high dose foetal weight, gestation rate and the number of foetuses were diminished due to an increased resorption rate. The slightly increased number of malformations observed in this group, were considered as spontaneous malformations which were not dose-dependent. The NOAEL for maternal toxicity was 100 mg/kg bw/day. The NOAEL for developmental toxicity was 100 mg/kg bw/day. No teratogenic effects were seen within this study.

The embryotoxic effects observed correlated with the marked maternal toxicity at the high dose and thus classification of Preventol KMX according to the dangerous substance directive 67/548/EEC and according to CLP 1272/2008/EC is not required.

Justification for classification or non-classification

Preventol KMX has not to be classified according to Dangerous substance directive 67/548/EEC and according to CLP (1272/2008/EC).