Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
A two-generation reproduction study with monochlorobenzene vapor in rats
Author:
Nair RS, Barter JA, Schroeder RE, Knezevich A & Stack CR
Year:
1987
Bibliographic source:
Fundamental and Applied Toxicology, 9:678-686
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
F1 generation was exposed to test substance 1 week after weaning instead of being exposed at weaning. Sperm parameters oestrus cycle not examined,. only brain, liver and kidney were weighed,reproductive organs (m+f), liver, kidneys microscopically
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): monochlorobenzene (MCB)
- Physical state: liquid
- Analytical purity: 99.9 %
-Boiling point: 132.1°C

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI
- Age at study initiation: (P) 6 wks; (F1) 6 wks
- Weight at study initiation: Males: 129-233 g; Females: 131-162 g
- Housing: all animals were individually housed in suspended stainless stell cages with wire mesh floors except during lactation.
- Diet (e.g. ad libitum): Rodent Laboratory Chow/5002 (registered trademark of Ralston Purina Company, St. Louis, MO) ad libitum.
- Water (e.g. ad libitum) :ad libitum during non-exposure period.
- Acclimation period: 13 days prior to initiation of exposure during non-exposure period.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 65-77°F
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12hrs dark/12hrs light cycle.


Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: preconditioned air
Details on exposure:
For generating an atmosphere of MCB vapor the test material was fed into an atomizing nozzle via an FMI fluid metering pump. The vaporized test material was diluted with preconditioned air prior to entry into the exposure chamber
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 10 days.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy.
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility of the same treatment group (random selection)
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Once the female had mated, she was housed individually for the remainder of the gestation period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
MIRAN 1 A organic vapour analyzer was used. At least 4 samples were drawn daily at hourly intervals from each exposure chamber.
Duration of treatment / exposure:
Premating exposure period (males): F0 males: 10 weeks; F1 males: 11 w eeks
Premating exposure period (females): F0 females: 10 weeks; F1 females: 11 w eeks
Exposure period: F0 and F1 generation: during mating period, d0 - 20 of gestation and d4 - 21 of lactation
Duration of test: until weaning (day 21 of lactation) of F2 pups
Frequency of treatment:
6 hour per day, 7 days per week
Details on study schedule:
Groups of 30 males and 30 females CD rats, designed as P generation, were exposed to vapor of monochlorobenzene (MCB) at target concentrations of 0 , 50 , 150, or 450 ppm (0, 234, 702 or 2106 mg/m³) for 10 weeks prior to mating and during mating, gestation, and lactation. The progeny of the P generation was designed as the F1 generation and groups of 30 male and 30 female F1 animals were exposed to the same concentration of MCB as the F0 parents. Exposure of F1 animals was initiated 1 week postweaning and lasted 11 weeks prior to the mating and through mating, gestation, and lactation. All F2 pups were observed through weaning at which time they were killed.
Observations made during the study included body weights, food consumption, mating and fertility indices, pup and litter survival indices, and histopathology of selected tissues.
Doses / concentrations
Remarks:
Doses / Concentrations:
F0 and F1 generation: 0, 50, 150 or 450 ppm (0, 234, 702 or 2106 mg/m³)
Basis:

No. of animals per sex per dose:
30
Control animals:
yes, concurrent vehicle
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All adult and weanling animals were observed for mortality and clinical signs twice daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were performed weekly.


BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded weekly; female body weights were recorded weekly prior to mating, on days 0, 4, 14, and 20 of gestation, on days 0, 4, 7, 14 and 21 of lactation, and weekly again following lactation.




Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
not tested
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible).


PARAMETERS EXAMINED
The following parameters were examined in [F0 / F1/ F2] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities.


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after weaning of the F1 generation.
- Maternal animals: All surviving animals were sacrificed after weaning of the F1 generation.




HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age (weaning).






Statistics:
Mean body weights, food consumption, organ weights, organ to body weight ratios, gestation lengths, and numbers of offspring were evaluated statistically using the following methods: Bartlett´s test, parametric methods (one-way analysis of variance which was followed by a Dunnett test) , Kruskal- Wallis test, and Dunn´s summed rank test.
Incidence data, which included pregnancy rates, fertility indices, mating indices, and litter survival indices, were analyzed using contingency tables.
First, a standard chi-square analysis was performed to determine of the proportion of indices differed between the groups tested. Next, each treatment group was compared to the control group using 2X2 Fisher exact test , and the significance level was corrected using the Bonferroni inequality. Also, an Armitage test for linear trend was performed.
Reproductive indices:
The mating index for males (ratio of number of males for which mating was confirmed in at least one female to total number of males) and
the mating index for females (ratio of number of females showing evidence of mating to total number of females),
pregnancy rate (ratio of number pregnant to number mated), and
fertlity index for males (ratio of number of males impregnating a female to number mating) were calculated for each of the two matings.
Offspring viability indices:
Pup viability index at birth (total number of live pups at day 0 / total number of pups observed (live plus dead pups at day 0 for individual females
Pup survival index at day 4: total number of live pups at day 4 (precull) / total number of live pups at day 0 for individual females
Pup survival index at day 21: total no of live pups at day 21 / total number of live pups at day 4 (postcull) for individual females
Litter survival index: total number of litters with live pups at weaning (day 21) / total number of litters with live pups at birth

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

REPRODUCTIVE PERFORMANCE
TEST DATA
Mating and fertility indices for males and females for both generations appeared unaffected by treatment
--Mean number of days to mate (control, 50, 150, 450 ppm)
P: 4.2, 3.1, 2.8, 3.1 F1: 4.9, 3.2, 3.1, 4.6
--Mating indices (%, females): control, 50, 150, 450 ppm
P: 100, 100, 96.7, 100 F1: 100, 96.7, 96.7, 93.3
--Mating indices (%, males): control, 50, 150, 450 ppm
P: 86.7, 93.3, 96.7, 96.7 F1: 90, 96.7, 96.7, 83.3
--Fertility indices , (%, males): control, 50, 150, 450 ppm
P: 92.3, 100, 93.1, 89.7 F1: 77.8, 100, 79.3, 88
--Pregnancy indices (%, females), control 50, 150, 450 ppm)
P: 90, 100, 93.1, 86.7 F1: 80, 100, 79.3, 89.3

HISTORICAL CONTROL DATA - CD rat (Bio/dynamics, Inc., 1978-1984)
represents reproduction indices for a total of 16 studies containing a total of 62 litter intervals:
------mean and range [....] of study values
--male mating index: mean = 92.6 % [66.7 % to 100.0 %]
--female mating index: mean = 88.3 % [70.0 % to 100.0 %]
--pregnancy rate:..........mean = 85.0 % [67.9 % to 100.0 %]
--male fertility index:....mean = 94.8 % [80.0 % to 100.0 %]

ORGAN WEIGHTS (PARENTAL ANIMALS)
Significant increases in mean absolute and/or relative liver weight were observed in the both P+F1 generation in the 150 and 450 ppm groups.
In the low-concentration group, only the relative liver weights of the F1 male rats were elevated.
males
absolute liver weights(g): control, 50, 150, 450 ppm
P: 19.25, 19.03, 21.48(p<=0.01), 21.84(p<=0.01) F1: 18.31, 19.46, 21.71(p<=0.01), 23.35(p<=0.01)
relative liver weights
P: 3.61, 3.60, 4.06(p<=0.01), 4.12(p<=0.01) F1: 3.47, 3.73(p<=0.05), 4.15(p<=0.01), 4.44(p<=0.01)
females
absolute liver weights(g): control, 50, 150, 450 ppm
P 11.45, 12, 12.08, 13.27(p<=0.01) F1: 12.36,12.7, 13.13, 13.95(p<=0.01)
relative liver weights
P: 3.77, 3.89, 3.95(p<=0.05), 4.41(p<=0.01) F1: 4.16, 4.17, 4.36, 4.59 (p<=0.01)

GROSS PATHOLOGY (PARENTAL ANIMALS)
--male, Increase in the incidence of small flaccid testes (50, 150, 450 ppm):
P: 0/30, 1/30, 3/30 F1: 0/30, 1/30, 5/30
--increase in incidence of dilated renal pelvis (control, 50, 150, 450 ppm)
male
P: 1/30, 1/30, 2/30, 4/30 F1: 1/30, 4/30, 6/30, 4/30
female:
P: 5/30, 4/30, 6/30, 5/30 F1: 0/30, 1/30, 2/30, 2/30

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic changes related to treatment were observed in the liver and kidney.

--Hepatocellular hypertrophy- incidence (0, 50, 150, 450 ppm)
male
P: 0/30, 0/30, 5/30, 14/30 F1: 2/30, 0/30, 3/30, 7/30.
All affected male rats showed only minimal to mild hepatocellular hypertrophy.
female
Only one high-concentration level female rat in the P generation showed hepatocellular hypertrophy (data not shown).

--Renal degeneration and inflammatory lesions were limited to the male rats.
The incidence and severity of renal changes were elevated in the mid-and high-concentration groups.
P. 30 males examined from each group: (0, 50, 150, 450 ppm)
bilateral tubular dilation/eosinophilic material : 0, 1, 4, 15
bilateral chronic interstitial nephritis : 1, 2, 7, 9
bilateral foci if regenerative epithelium : 0, 1, 5, 8
P: 30 females were examined of the control and 450 ppm group:
bilateral chronic interstitiial nephritis 0/30; 2/30
F1. 30 males examined from each group: (0, 50, 150, 450 ppm)
bilateral tubular dilation/eosinophilic material : 4, 3, 8, 16
bilateral chronic interstitial nephritis : 0, 1, 6, 11
bilateral foci if regenerative epithelium : 1, 0, 4, 10
F1: 30 females were examined of the control and the 450 ppm group
Unilateral tubular dilatation /eosinophilic material: 0/30; 3/30

Effect levels (P0)

open allclose all
Dose descriptor:
other: NOAEC (fertility)
Effect level:
ca. 450 ppm
Sex:
male/female
Basis for effect level:
other: Monochlorobenzene did not have any adverse effects on reproductive performance or fertility through 2 consecutive generations
Remarks on result:
other: Generation: P, F1 (migrated information)
Dose descriptor:
other: NOAEC (developmental)
Effect level:
ca. 450 ppm
Sex:
male/female
Basis for effect level:
other: Pub survival indices were considered comparable for control and treated animals in both generations
Remarks on result:
other: Generation: F1, F2 (migrated information)
Dose descriptor:
other: LOAEC (systemic toxicity)
Effect level:
ca. 50 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: P, F1 (migrated information)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING)
No mortality was observed in the treated and control groups of the F1 generation.
In F2 litters, a slight decrease in pup survival index (days 0-4) was observed in the higest concentration level only.
The majority of the decrease in pup survival index(day 0-4) at the highest concentration level was attributed to two dams; one dam lost 12 of 15 pups during lactation days 0-4, and the other dam lost all 10 pups during the same time period. The authors stated that excluding pup survival data for these 2 litters the day 0-4 pup survival index for the high-concentration group was94.5 % which is similar to control value. they concluded that if this had been a compound-related response, more affected litters would have been expected.
Therefore, pup survival indices for control and treated animals for both litters were considered comparable.

--Pup viability index [%] (0, 50, 150, 450 ppm)
F1: 96.9, 96.1, 99, 96.4 F2: 98.1, 97.2, 97.9, 97.4

--Pup survival index [%] day 0-4 (0, 50, 150, 450 ppm)
F1 92.4, 96.7, 94.1, 90.5 F2: 94.4, 93.2, 97.6, 87.7

--Pup survival index [%] day4-21 (0, 50, 150, 450 ppm)
F1: 96.7, 95, 97.1., 93.5 F2: 88.4, 99.1, 89.1, 89.6
--Litter survival index [%] (0, 50, 150, 450 ppm)
F1: 100, 96.7, 96.3, 92.3 F2: 91.7, 96.6, 91.3, 88

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

F0 mating (for F1 litters)

Group (ppm)              Pregnancy, female (percentage)       Fertility, males

0                        90.0                                   92.3

50                       100.0                                  100.0

150                      93.1                                   93.1

450                      86.7                                   89.7

F1 mating (for F2 litters)

Group (ppm)              Pregnancy, female (percentage)       Fertility, males

0                        90.0                                   77.8

50                       96.7                                  100.0

150                      96.7                                   79.3

450                      83.3                                   88.0

Applicant's summary and conclusion

Executive summary:

Groups of 30 males and 30 females CD rats, designed as P generation, were exposed to vapor of monochlorobenzene (MCB) at target concentrations of 0 , 50 , 150, or 450 ppm (0, 234, 702 or 2106 mg/m³) for 10 weeks prior to mating and during mating, gestation, and lactation. The progeny of the P generation was designed as the F1 generation and groups of 30 male and 30 female F1 animals were exposed to the same concentration of MCB as the F0 parents. Exposure of F1 animals was initiated 1 week postweaning and lasted 11 weeks prior to the mating and through mating, gestation, and lactation. All F2 pups were observed through weaning at which time they were killed.

Observations made during the study included body weights, food consumption, mating and fertility indices, pup and litter survival indices, and histopathology of selected tissues.

No mortality was observed during the course of this study. No adverse effect of treatment was evident on body weight, food consumption, and physical observation. Mating and fertility indices for males and females for both generations appeared unaffected by treatment.The slightly reduced pregnancy indices mainly at the highest concentration level (0, 50, 150, 450 ppm: P: 90, 100, 93.1, 86.7 F1: 80, 100, 79.3, 89.3) were within the historical control data of the testing facility in the respective time interval (CD rat at Bio/dynamics, Inc., 1978 -1984, representing pregnancy rates for a total of 16 studies containing a total of 62 litter intervals:

mean = 85.0 % [67.9 % to 100.0 %]. Body weights and food consumption for all treated groups were comparable to controls during the growth period. Maternal body weight data during gestation and lactation were also comparable between the control and treated groups

Overall, reproductive performance, fertility and development of pups were not affected by treatment with monochlorobenzene yielding a NOAEC of 450 ppm (corresponding to 2106 mg/m³) for reproduction and development.

However, post mortem examinations revealed significantly elevated absolute and relative liver weights in F0 and F1 male and female rats at 150 and 450 ppm and at 50 ppm in F1 males accompanied by hepatocellular hypertrophy in F0 and F1 males exposed to 150 and 450 ppm. In addition, renal changes (tubular dilation with eosinophilic material, interstitial nephritis, and foci of regenerative epithelium) were observed mainly in F0 and F1 male rats exposed to 150 and 450 ppm. The significance of reported degeneration of the testicular germinal epithelium in male rats at 450 ppm is unclear because no histopathological correlate is reported. Thus, the LOAEC (systemic toxicity) is considered to be 50 ppm (corresponding to 234 mg/m³).