Pitch, coal tar, high-temp.

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Studies on reproductive toxicity using pitch, coal tar, high temp. are not available. In a fertility study with benzo[a]pyrene following no guideline (exposure of pregnant dams from day 7 to day 16 of gestation, monitoring of reproductive success and development of F1 offspring and reproductive performance of F1 males and females) adverse effects on fertility were observed even at the lowest concentration tested (10 mg/kg bw/d).

Link to relevant study records

Reference

Endpoint:

reproductive toxicity, other

Remarks:

Investigation of development and reproductive function of untreated F1 males and females originating from dames treated during day 7 to 16 of gestation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study meets basic scientific principles; basic data given; acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Pregnant mice were treated with graduate doses of benzo[a]pyrene from day 7 through day 16 of gestation. Litters were monitored with regard to viability of litter, litter size, and mean pup weight development from parturition up to day 42 of life. At 7 or 8 weeks of age, F1 animals treated prenatally were introduced into a breeding study. Treated males were mated with untreated females and treated females with untreated males, respectively. Reproductive performance was examined for five mating periods (treated males) and for a 6 month mating period (treated females).

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): benzo(a)pyrene, BP
- Source: Aldrich Chemical Co., Inc., Milwaukee, WI, USA
- Analytical purity: no data

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Labs. Inc., Wilmington, MA
- Age at study initiation: adult animals (P); 7 to 8 weeks (F1, breeding study)
- Weight at study initiation: mean pup weight (F1) at day 42 post partum 26.8 to 29.9 g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): Purina lab chow, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 1 week

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
- suspension in vehicle (corn oil)

VEHICLE
- Amount of vehicle (if gavage): 0.2 ml

Details on mating procedure:

PARENTAL MATING
- M/F ratio per cage: 1/3 (proven breeder males with mice that had at least one litter)
- Length of cohabitation: no data

MATING OF F1 GENERATION (males treated before in utero)
- M/F ratio per cage: 1/2
- Length of cohabitation: 5 d
- Proof of pregnancy: vaginal plug referred to as day 1 of pregnancy
- Further matings: yes; as general approach, mating was replicated five times each with two new virgin mice
- After successful mating each pregnant female was caged (how): no data
- Any other deviations from standard protocol: pregnant females were allowed to deliver and fertility parameters were recorded

MATING OF F1 GENERATION (females treated before in utero)
- M/F ratio per cage: 1/1 (female housed continuously with one proven breeder male)
- Length of cohabitation: 6 month
- Proof of pregnancy: vaginal plug referred to as day 1 of pregnancy
- After 30 days of unsuccessful pairing replacement of first male by another male with proven fertility
- After successful mating each pregnant female was caged (how): no data
- Any other deviations from standard protocol: pregnant females were allowed to deliver and fertility parameters were recorded

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Pregnant mice of P generation were treated from day 7 through day 16 of gestation.
P generation males and F1 generation animals (males and females) did not get any treatment.

Frequency of treatment:

daily

Details on study schedule:

- F1 animals (males and females, exposed to BaP in utero (treatment of P generation females) for 10 days) were mated at the age of 7 to 8 weeks with untreated mice of the opposite sex. F1 animals not selected for the breeding study were sacrificed.

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

160 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Parental animals (only females treated)
Dose 0 10 40 160 mg/kg bw/day
females 60 30 60 30
F1 animals (breeding study)
males 45 25 45 20 (exposure only in utero) (mated with untreated females)
females 35 35 55 20 (exposure only in utero) (mated with untreated male)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on preliminary study

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: no data

DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: no data

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

Parameters examined in F1 male generation:
testis weight (10 animals)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- 8 pups/litter, 4 of each sex as nearly as possible; excess pups were killed and discarded

PARAMETERS EXAMINED
- The following parameters were examined in P offspring:
- number of pups, presence of gross anomalies (day 1); sex and weight of pups (day 4); viable litters, mean litter size, mean pup weight (day 4, 20, 42 post partum)
- The following parameters were examined in F1 offspring:
- number of pups, presence of gross anomalies (day 1); sex and weight of pups (day 4); weight and gross examination (day 20 post partum)

GROSS EXAMINATION OF DEAD PUPS:
- no data

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: no data
- Maternal animals: day 20 post partum

GROSS NECROPSY:
- No examination

HISTOPATHOLOGY / ORGAN WEIGHTS
- No examination

Postmortem examinations (offspring):

ANIMALS NOT USED FOR BREEDING
- The P offspring not selected as F1 parental animals were sacrificed at 7 to 8 weeks of age. F1 offspring was sacrificed on day 20 post partum. No postmortem examinations (macroscopic and/or microscopic examinations) were conducted for these animals with the exception given below.
- From the 0, 10, and 40 mg BaP exposure group, 10 animals of either sex were sacrificed at 6 weeks of age and gross necropsy was performed.

MALE BREEDING STUDY
- No information on males
- Pregnant females were sacrificed 14 days after separation from the males (days 14-19 of gestation). Number of implants, foetuses, and resorptions were recorded

FEMALE BREEDING STUDY
- Females were left with a proven breeder male until the conclusion of the study. No postmortem examination of dams is recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
- 10 F1 male and female mice exposed to 0, 10, and 40 mg B(a)P/kg bw/day (treatment of parental females from day 7 through 16 of gestation) were subject to gross necropsy at 6 weeks of age (see above).
- Reproductive tissues were removed, trimmed, weighed (testes only), fixed in alcohol-formalin-acetic acid (AFA), sectioned, stained with Harris's hematoxylin and eosin, and prepared for microscopic examination.
- The gonads of each animal were sectioned transversely through the longest diameter of the organ. As observed lesions were approximately equal in number and uniformly distributed throughout the section, additional serial sections were deemed unnecessary.

Statistics:

Data on pregnancy maintenance and foetal survival were analysed for statistical significance by appropriate nonparametric procedures (Siegel S. 1956, Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill Book Co., New York).
The least significant difference method was used to analyse data on litter size and foetal and testes weights (Steel and Torrie 1960, Principle and Procedures of Statistics. McGraw-Hill Book Co., New York).

Reproductive indices:

Fertility index: Females pregnant / females exposed to males x 100

Clinical signs:

not examined

Mortality:

not specified

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In the highest dose group (160 mg/kg bw/day), percentage of pregnant females was significantly reduced (from ca. 75 to 50 %).

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: only pregnant females were treated in this study (day 7 to 16 of gestation)

Key result

Dose descriptor:

LOAEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: only pregnant females were treated in this study (day 7 to 16 of gestation)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

160 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

not specified

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

VIABILITY (OFFSPRING)
Viability of the P generation offspring (F1 pups) was significantly reduced in the highest dose group (160 mg/kg bw/day) (about 40 to 50% - see table 1 below).

CLINICAL SIGNS (OFFSPRING)
No data on clinical observation of animals are reported

BODY WEIGHT (OFFSPRING)
At day 20 and 42 post partum, mean pup weight of F1 animals was significantly reduced (see table 1 below)

SEXUAL MATURATION (OFFSPRING)
Gonads of F1 animals were strongly impaired (reduced size and function) even at the lowest dose.

ORGAN WEIGHTS (OFFSPRING)
Testes weights of F1 males were significantly reduced already in the lowest dose group (by 40%).

GROSS PATHOLOGY (OFFSPRING)
No data reported

HISTOPATHOLOGY (OFFSPRING)
In males, increasing testicular damage was seen with increasing doses. Females exposed to BaP had no ovaries or only remnants of ovarian tissues (see below)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Sex:

male/female

Basis for effect level:

sexual maturation

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

not determinable

Remarks:

a NOAEL could not be identified; adverse effects were already seen at the lowest dose tested

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects in F1 generation animals: body weight at day 42 post partum; organ weights (gonads) of males and females; histopathology of gonads; fertility index in the breeding studies with F1 animals

Remarks on result:

other: lowest dose tested

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 other: mg/kg bw/day (nominal) (lowest dose tested)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 other: mg/kg bw/day (nominal) (lowest dose tested)

System:

female reproductive system

Organ:

ovary

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

10 other: mg/kg bw/day (nominal) (lowest dose tested)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Effects of maternal treatment from day 7 to 16 of gestation on pregnancy and F1 offspring is displayed in the following table

	Benzo[a]pyrene   [mg/kg bw/day]
Dose	0 (control)	10	40	160
Females treated	60	30	60	30
Females pregnant (%)	46 (77)	22 (73)	46 (77)	15 (50)*
Parturition examination
Viable litters (%)	46 (77)	21 (70)	44 (73)	13 (43)**
Mean litter size^a	9.4 ± 0.6	8.6 ± 0.8	10.5 ± 0.5	8.8 ± 0.9
4 Day examination
Viable litters	45	20	42	12
Mean litter size^a	9.5 ± 0.6	8.6 ± 0.7	10.5 ± 0.5	8.3 ± 1.0
Mean pup weight [g]	2.7 ± 0.02	2.8 ± 0.04	2.5 ± 0.02	2.2 ± 0.04
20 Day examination
Viable litters (%)	45 (75)	20 (67)	40 (67)	10 (33)**
Mean litter size^a/b	7.1 ± 0.3	7.0 ± 0.5	7.5 ± 0.2	6.5 ± 0.6
Mean pup weight [g]	11.2 ± 0.1	11.6 ± 0.1	10.4 ± 0.1**	9.7 ± 0.2**
42 Day examination
Viable litters (%)	45 (75)	20 (67)	40 (67)	10 (33)**
Mean litter size^a/b	7.0 ± 0.3	6.8 ± 0.5	7.1 ± 0.2	5.9 ± 0.5
Mean pup weight [g]	29.9 ± 0.2	28.2 ± 0.3**	28.0 ± 0.2**	26.8 ± 0.4**

^aMean number of live pups per viable litter ± SEM

^bAll litters were standardised t 8 pups per litter on Day 4

*Significantly different from controls (P<0.05)

**Significantly different from controls (P<0.01)

BaP was not toxic to mothers or foetuses at any dose tested. All litters appeared normal by gross observation. But pregnancy maintenance was reduced by ca. 25% at the 160 mg/kg bw/day dose level, and there was a significant decrease at 20 and 42 days of age in weights of pups from treated dams.

 

Reproductive performance of male and female F1 mice exposed prenatally to BaP (from day 7 through day 16 of gestation) (mean ± SEM)

	Benzo[a]pyrene   [mg/kg bw/day]
Dose	0 (control)	10	40	160
Male breeding study
Number F1 males tested^a (number of fertile males)	45 (45)	25 (20)	45 (3)	20 (0)
Fertility index^b	80.4	52.0*	4.7**	0.0**
Mean litter size	11.0 ± 0.1	10.7 ± 0.2	10.8 ± 0.6	---
Female breeding study
Number F1 females tested^c	35	35	55	20
Fertility index^b	100.0	65.7**	0.0**	0.0**
Mean litter size	12.9 ± 0.2	10.4 ± 0.4**	---	---

 

^aBeginning at 7 weeks of age, each F1 male was exposed to 10 untreated females over a period of 25 days.

^bFertility index: Females pregnant / females exposed to males x 100

^cBeginning at 6 weeks of age, each F1 female was cohabitated continuously with an untreated male for a period of 6 month.

*Significantly different from controls (P<0.05)

**Significantly different from controls (P<0.01)

Adverse effects on reproduction of the F1 animals (male and female breeding studies) were seen at all dose levels increasing with dose. At the highest dose (160 mg/kg bw/day), all of the male mice were infertile and no litter was produced. For females, animals of the two highest dose groups (40 and 160 mg/kg bw/day) did not generate any offspring. At the lowest dose (10 mg/kg bw/day), fertility and ability to produce offspring was already reduced by ca. 20 to 30%.

There were no gross abnormalities in F2 offspring from the F1 reproduction studies, and there were no significant differences among treatment groups in F2 pup mean body weights at 4 and 20 days of age in the female breeding study.

The most obvious effect of prenatal exposure to BaP was a dramatic decrease in the size of the gonads. Paired testes weights were significantly reduced, and testes from animals exposed in utero to 10 and 40 mg BaP weighed only ca. 60% and 18% of the testes weights from control animals, respectively.

Histologic examination of gonadal tissue sections revealed significant alterations following exposure to BaP as well. In males, increasing testicular damage was seen with increasing doses. In the low dose group (10 mg/kg bw/day), testes of all males showed evidence of tubular injury, but each animal also had some seminiferous tubules that displayed active spermatogenesis. In the 40 mg/kg bw/day group, tubules were smaller compared to control animals and they were empty except for a basal layer of cells. Females exposed to BaP had no ovaries or only remnants of ovarian tissues. Ovarian tissue was hyperplastic with very few follicles and corpora lutea. At higher doses (40 mg/kg bw/day) no evidence of folliculogenesis could be observed. Ovarian tissue fragments consisted of lutein cells, medullary cords, and very little interstitial tissue.

Conclusions:

Adverse effects on reproduction of the F1 animals (male and female breeding studies) were seen at all dose levels increasing with dose. Therefore, a NOAEL could not be derived. The LOAEL was determined to be 10 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Pitch, coal tar, high-temp. (CTPht) is a genotoxic carcinogen with adequate risk management measures implemented. Thus according to REACH regulation, studies on reproductive toxicity are not required.

Benzo[a]pyrene is present in CTPht in a concentration up to 1.5 %. In the absence of data for CTPht itself, reproductive effects of BaP may be used to characterise the reproductive potency of pitch, coal tar, high-temp.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

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

Pitch, coal tar, high-temp. is a genotoxic carcinogen with adequate risk management measures implemented. Thus according to REACH regulation, studies on developmental toxicity are not required.

Justification for classification or non-classification

Benzo[a]pyrene is classified as reproductive toxicant Cat. 1B; based on a content of up to 1.5 % of benzo[a]pyrene, pitch, coal tar, high-temp. is self-classified by the registrant as reproductive toxicant Cat. 1B. This is in accordance with the classification of pitch, coal tar, high-temp. in Annex VI, Part 3, Table 3 of Regulation (EC) 1272/2008 (CLP regulation, including amendments) as Repr. 1B.