Registration Dossier

Administrative data

Endpoint:
multi-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Near guideline study. Published in peer reviewed literature. Minor restrictions in design and/or reporting but otherwise adequate for assessment.

Data source

Reference
Reference Type:
publication
Title:
The reproductive and developmental toxicity of high flash aromatic naphtha
Author:
McKee RH, Wong ZA, Schmitt S, Beatty P, Swanson M, Schreiner CA and Schardein JL
Year:
1990
Bibliographic source:
Toxicology and Industrial Health Vol 6, No. 314, pp 441-460

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Pre 2001 Guideline.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Test sample conformed to specifications for High-Flash Aromatic Naptha, Type 1 (as defined by ASTM method D-3734. Although commercially available, the test sample was specially prepared to meet the compositional requirements (i.e. at least 22% ethyltoluene isomers, at least 15% trimethylbenzene isomers and at least 75% total ethyltoluenes and trimethylbenzenes) specified in the test rule.

The sample was analysed for the weight percent reported as follows:
o-xylene: 3.20
cumene: 2.74
n-propylbenzene: 3.97
4-ethyltoluene: 7.05
3-ethyltoluene: 15.1
2-ethyltoluene: 5.44
1,3,5-trimethylbenzene: 8.37
1,2,4-trimethylbenzene: 40.5
1,2,3-trimethylbenzene: 6.18
>=C10's: 6.19
TOTAL: 98.74

Test animals

Species:
rat
Strain:
other: Charles River COBS CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI, USA
- Age at study initiation: F0, 8-9 weeks old; F1, 5-7 weeks old; F2, 5-7 weeks old; F3, 22 days old
- Weight at study initiation: not reported
- Fasting period before study: none
- Housing: individually housed in wire mesh cages throughout the study, except during mating period when males and females co-habited (1:1).
- Housing during parturition: between gestational day (GD) 20 and lactational day (LD) 4, pregnant dams were individually housed in plastic cages with wood chips supplied for nesting materials.
- Diet: Purina Certified Rodent Chow No. 5002 ad libitum except during exposures
- Water : provided ad libitum - no other details
- Acclimation period: parents - 2-3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported

IN-LIFE DATES: not reported

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 16 cubic metre glass and stainless steel chambers
- Method of holding animals in test chamber: not specified
- Source and rate of air: chamber air supplied by HVAC system, separate from general laboratory air handling system
- Method of conditioning air: HVAC system, particulate-filtered and controlled for temperature and humidity.
- System of generating particulates/aerosols: test sample was vaporised with nitrogen through a heated glass column. Vapours diluted with chamber ventilation air to achieve desired exposure levels
- Temperature, humidity, pressure in air chamber: monitored every 30 minutes
- Air flow rate: not reported
- Air change rate: not reported
- Treatment of exhaust air: not reported

TEST ATMOSPHERE
- Brief description of analytical method used: gas-phase infrared spectrometer
- Samples taken from breathing zone: not specified
Details on mating procedure:
30 males and females per group were co-housed (1:1) for a 2 week mating period from each of the 3 generations.
F2 parents: due to toxicity, all surviving animals in the high dose group were mated, to produce the F3 generation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure levels were monitored with a gas-phase infrared spectrophotometer at hourly intervals throughout the study. The accuracy of this method was confirmed by the use of vapour standards. Additionally, the composition (on a weight percentage basis) of the test material within the exposure chamber was determined by gas chromatography during the first exposure week.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
F0 males: 6 hours per day, 5 days per week for 10 weeks before mating and 5 days/week during the 2 week mating period.
F0 females: 6 hours per day, 5 days per week for 10 weeks before mating, 5 days per week during mating, daily during pregnancy (gestational day 0 to 20) and daily from postnatal day 5 (LD5) until weaning (LD 21).
F1 males : no exposure after weaning until 5-7 weeks of age then 6 hours per day, 5 days per week for 10 weeks before mating and 5 days/week during the 2 week mating period.
F1 females: no exposure after weaning until 5-7 weeks of age then 6 hours per day, 5 days per week for 10 weeks before mating, 5 days/week during mating, daily during pregnancy (gestational day 0 to 20) and daily from postnatal day 5 (LD5) until weaning (LD 21).
F2 males : From postnatal day 22, 6 hours per day, 5 days per week for 10 weeks before mating and 5 days/week during the 2 week mating period.
F2 females: From postnatal day 22, 6 hours per day, 5 days per week for 10 weeks before mating, 5 days/week during mating, daily during pregnancy (gestational day 0 to 20) and daily from postnatal day 5 (LD5) until weaning (LD 21).
Details on study schedule:
- Age at start of 10 week exposure period: F0, 8-9 weeks; F1, 5-7 weeks; F2, 22 days.
- Random selection of parents from F1 generation (30/sex/group) was done approximately 1 week after all had completed weaning on LD21.
The study was extended to a 3rd generation but exposure of the F2 animals used to produce the F3 generation began immediately after weaning (LD 22). Due to concerns about toxicity, 40 animals/sex/group were initially randomised and exposed. 30 animals/sex/group were then randomly selected for mating in the control, 100 and 500ppm groups. Due to the very high number of deaths in the first week at 1500ppm, all surviving animals were mated to produce the F3 pups.
- Age at mating of the mated animals in the study: F0, 18-19 weeks; F1, 15-17 weeks; F2, 14 weeks .
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 100, 500 and 1500 ppm
Basis:
other: target concentrations
Remarks:
Doses / Concentrations:
0, 103 ± 2.1,495 ± 8.0, 1480 ± 20.5 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0, 107±2.4, 513±12.8 and 1483±33.0 ppm
Basis:
other: nominal concentration
No. of animals per sex per dose:
Exposure groups: 3 groups plus control group.

F0: 30 males caged with 30 females/group;
F1: 30 males caged with 30 females /group;
F2: 30 males caged with 30 females in control, low and mid dose groups, all survivors (4 males and 6 females) mated in high dose group.
Control animals:
other: exposed to air only
Details on study design:
- Dose selection rationale: dose levels based on previous developmental and reproductive toxicity studies in rats of Aromatol, a branded product conforming to the specifications of High Flash Aromatic Naphtha.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: recorded weekly until confirmation of mating
Females were also weighed on gestational days 0, 7, 14 and 21 and lactational days 0, 7, 14 and 21

FOOD CONSUMPTION: Yes
- Food consumption was measured weekly except during mating, gestation and lactation

Oestrous cyclicity (parental animals):
No information
Sperm parameters (parental animals):
No information
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); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 / F3 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain.

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 at the end of the mating period.
- Maternal animals: All surviving animals after weaning.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: epididymides, lung, ovary, testis, prostate/seminal vesicle and uterus/vagina.
The following organs and tissues were submitted: epididymides, lung, ovary, pituitary, prostate, seminal vesicle, testis, uterus, vagina and regional lymph nodes and were examined microscopically in the control and high dose groups only. Any masses and gross lesions in any groups were examined.
Postmortem examinations (offspring):
SACRIFICE
- The F1 and F2 offspring not selected as parental animals were sacrificed at 4 days of age.
- These animals and any that died during the lactation period were necropsied and examined for anomalies.

GROSS NECROPSY
- Gross necropsy for anomalies - no further details

Statistics:
Fertility indices, sex ratio: Chi-square test criterion with Yates' correction for 2x2 contingency tables.
Parental body weights, maternal body weight changes, organ weights: analysis of variance (one-way), Bartlett's test for homogeneity of variance and appropriate t-test (for equal or unequal variance) using Dunnett's multiple comparison tables.
Mean no liveborn pups/litter, mean pup weight: analysis of variance and appropriate t-test.
Reproductive indices:
Female mating index, female conception index, female gestation index, male fertility index, cohabitation time required for mating, litter size at birth.
Offspring viability indices:
Gestation survival index, postnatal survival index for days 4 and 21.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
F0 females - at 1500 ppm, 7 females died or were killed - 3 prior to mating, 3 during gestation and 1 during lactation.
F1 males & females - at 1500 ppm ataxia was seen in 18 males and 23 females and reduced motor activity was seen in 11 males and 8 females.
F1 females - at 1500 ppm, 6 females died - 3 during exposure, 1 during gestation, 1 during delivery and 1 on day 2 of lactation. One female died in each of the other 3 groups.
F2 males & females - exposures were initiated on day 22 and most animals at 1500 ppm (36/40 males and 34/40 females) died within the first week.
BODY WEIGHT (PARENTAL ANIMALS)
F0 males - body weight gain was significantly reduced at 500 ppm (by (5-7%) and at 1500 ppm (by 14-16%).
F0 females- body weight gain was significantly reduced at 500 ppm (by (5-7%) and at 1500 ppm (by 5-7%).
F1 males & females - mean body weights were less than controls (by 17% and 12%, respectively) at the start of exposure. During exposure, bodyweight gain was reduced by 5-7% in males at 500 ppm, 21-25% in males at 1500 ppm and 9-14% in females at 1500 ppm.
F2 males & females - at 1500 ppm, body weights of survivors were 40% & 35% (males/females) below controls in week 4 and remained 31-38% / 21-30% (males/females) below controls for the remainder of the exposure period.
F2 males& females - at 500 and 100 ppm, body weights were initially lower than controls and remained at similar levels throughout the exposure period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no effects on food consumption at any exposure level in any of the generations.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Male fertility was statistically significantly lower (p< 0.05) at the 1500ppm group in the F1 generation only.

Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
500 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Based on severe systemic toxicity at higher dose level. No consistent evidence of reproductive toxicity in the presence of systemic toxicity
Remarks on result:
other: Generation: F0, F1, F2, F3 (migrated information)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING)
F2 - the mean number of live offspring/litter and number of live births/number delivered were reduced in the 1500 ppm group but this was associated with unconfirmed matings. Where mating was not confirmed, exposure of the dams continued until delivery.
The fraction of liveborn offspring was slightly but significantly reduced in the high dose group but survival throughout the weaning period was similar to controls.
F3 - there was little evidence of reproductive toxicity even at 1500 ppm, even though there were only 6 litters for evaluation.

CLINICAL SIGNS (OFFSPRING)
No effects.

BODY WEIGHT (OFFSPRING)
F2 - mean body weights were lower in 1500 ppm group from LD7 throughout the rest of the weaning period.
F3- mean bodyweights at 1500 ppm were significantly lower at birth but there were no significant differences at LD4. The reduced birth weight may have been a result of the small size of the dams.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Exposure to analysed concentrations of 1500 ppm of High Flash Aromatic Naphtha caused toxicity but no consistent evidence of reproductive toxicity.
Executive summary:

In the first generation parental body weights were significantly reduced at 1500 ppm but reproductive parameters were unaffected. There were no differences in litter size, mean birth weight, or postnatal survival. Once maternal exposures were reinitiated (at day 4L), offspring in the 1500 ppm group gained weight more slowly than controls.

 

In the second generation parental body weight gain was again reduced at 1500 ppm and, male fertility was lower. Litter size, birth weight, and postnatal survival appeared to be reduced in the 1500 ppm exposure group although this was affected by the inclusion of several litters exposed beyond GD20, until just prior to birth. In these litters, the frequency of live births and mean birth weight was reduced and survival early in lactation was poor. Litters from dams exposed to 1500 ppm to GD20 pup survival was 92.5% compared with 98.7% in controls.

 

For the third generation, exposure of each rat was continuous (i.e. there was no exposure-free period between weaning and commencement of exposure as in previous generations). In the 1500 ppm group, 88% of the rats died. There was no evidence of fertility or reproductive effects although only 6 litters were available for evaluation at 1500 ppm. Birth weights were lower in the 1500 ppm group although mean body weight at LD4 was not different from control. The toxicity of 1500 ppm in this generation precludes definitive evaluation of the potential for reproductive effects and clarification of the results obtained in the previous generation.