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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-08-12 to 2004-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
1,2,3,4-tetrahydronaphthalene of Degussa AG, batch No. 2507330, produced 25 July 2003, purity >= 98.0 %

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ORGANISMS
- Source: Harlan Winkelmann, D-33178 Borchen
- Strain: Hsd: Sprague Dawley SD
- Age: 9-11 weeks
- Weight at study initiation: 208 g (mean)
- Number of animals: 24 per group
- Controls: vehicle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other:  sesame oil
Details on exposure:
ADMINISTRATION / EXPOSURE
- Vehicle: sesame oil
- Concentration in vehicle: 0; 3.75; 11.25; 33.75 mg/ml
- Total volume applied: 4 ml/kg bw/day, adjusted to most recently  recorded body weight
Details on mating procedure:
MATING PROCEDURES: Virgin females in the pre-oestrus or oestrus phase  were mated overnight with sexually mature males (ratio 1 male : 1 female)  and were caged individually after the detection of sperm in vaginal  smears. The day of sperm detection was defined as day 0 of gestation.  Pregnancy was confirmed at necropsy.
Duration of treatment / exposure:
days 6 through 19 of pregnancy (day 0 = sperm detection)
Frequency of treatment:
once daily
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: sacrifice on day 20

Examinations

Maternal examinations:
PARAMETERS ASSESSED DURING STUDY: 
- Body weights: days 0, 3, 6, 9, 13, 16, 18, 20
- Food consumption: for intervals between body weight determinations
- Clinical observations: survival, health condition and behavior twice  daily (once daily on weekends and public holidays); individual clinical 
observations once daily
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Macroscopic: P external and internal (thoracic and abdominal contents)  for macroscopically visible changes, with emphasis on the uterus; F1 for  
visible abnormalities
- Organ weights: gravid uterus weight with at least one fetus
Ovaries and uterine content:
- Examination of uterine content: Live and dead fetuses as well as  conceptuses undergoing resorption, placentas and corpora lutea were  counted, 
identified in numerical sequence from cervix to ovary and  examined macroscopically for visible abnormalities. The implantation  sites in the 
uterus were counted after staining with ammonium sulfide.
Fetal examinations:
- Examination of fetuses: Determination of weight, crown-rump length,  examination for gross external abnormalities.  Approximately half of the 
live fetuses of each litter were skinned and  fixed in alcohol, necropsied, sexed and checked for anomalies of the  internal organs (including 
particular attention to the reproductive tract  for signs of altered development). The carcasses were placed in a  solution of potassium hydroxide 
for clearing and stained with Alizarin  red S and Alcian blue (double-staining). The skeletons (bones and  cartilage) were examined and checked 
for stage of development and  abnormalities with the aid of a stereomicroscope. The remaining fetuses were transferred in Bouin's solution, 
necropsied,  sexed and examined for organ  anomalies (including particular attention  to the reproductive tract for signs of altered development) 
referring to  Wilson's slicing technique. Visceral and skeletal changes were subdivided into four categories (major  defects, minor defects, 
variations, and retardations) based on the  severity and / or the spontaneous incidence of the finding.
Statistics:
The statistical evaluation was based on the assumption of a monotonic dose-response relationship. A step-down trend test procedure was therefore applied, using all doses to determine the dose level at which there was no statistical significance of trend for the parameter. Statistical evaluations
over the low dose group(s) were only carried out if significant effects were detected in the higher dose group(s) (Hothorn & Lehmacher, 1991).
Maternal body weight was analyzed for statistical significance (p ≤ 0.05) using a 1-way ANOVAbased ordinal step-down trend test (see Tukey et al.,
1985). Food consumption was analyzed for statistical significance (p ≤ 0.05) using a Jonckheere test (Jonckheere, 1954; Lin & Haseman, 1975)
associated to the step-down ordinal test procedure.

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
135 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
135 mg/kg bw/day
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Result: not teratogenic
MATERNAL TOXIC EFFECTS BY DOSE LEVEL: 
- Mortality: There was no treatment-related death.
- Description, severity, time of onset and duration of clinical signs:  Clinical signs did not occur.
- Body weight: Slightly decreased in high dose group, statistically  significant (p<=0.05) on days 9 (-3%), 18 (-5%), and 20 (-5%).

A  significantly lower body weight gain was recorded for the whole treatment  period (0-20: -15%). Body weights slightly 

decreased also in the mid dose group with  statistical significance on days 9 (-11%) and 13 (-6%) and subsequent  adaptation to 

normal, thus considered to be of questionable biological  relevance. Normal in low dose group.
- Food/water consumption: Food consumption distinctly to slightly  decreased in high dose group, statistically significant on study days 
6-9: -33%; 9-13: -12%; 13-16: -10%; 16-18: -12% (absolute); 
6-9: -32%; 9-13: -10%; 13-16: -7%; 16-18: -9% (relative). 
Food consumption slightly to marginally lower in mid dose females,  attaining statistical significance on days 6-9 (-15% abs., 

-16% rel.) and  13-16 (-6% abs. and rel.) only. Normal in low dose group.
- Number pregnant per dose level: 22/24; 22/24; 23/24; 23/24 (control /  low / mid / high dose) = not affected. One control female 

was pregnant  with only corpora lutea and empty implantation sites
- Number aborting: no abortions
- Number of resorptions: no total resorptions
- Number of implantations: 13.6; 14.5; 13.6; 13.7 (control / low / mid /  high dose) = not affected. 
- Pre and post implantation loss: 4.9; 6.2; 8.3; 10.9% pre-implantation  loss (%corpora lutea) (means of control / low / mid / high dose). One  total post implantation loss in control group.
- Number of corpora lutea: 14.3; 15.5; 14.6; 15.4 (means of control / low  / mid / high dose).
- Gross pathology incidence and severity: No compound-related gross  lesions were observed at necropsy.
- Organ weight changes: Insignificantly lower uterus weight for high dose  females (-7%).

FETAL DATA: 
There were no findings at caesarian section in any group of fetuses which  could be related to the test substance administration. 
There was no statistically significant difference  in the mean crown-rump  length for either male or female fetuses in any group. 

However,  evaluation of both genders together revealed a slight but statistically  significant (p<=0.05) decrease of the mean 

crown-rump length for all high  dose fetuses against the control (-2.3%). 
The mean placenta weight was slightly but statistically significantly  (p<=0.05) decreased in the high dose group 

(0.52; 0.51; 0.51; 0.46 g in  control, low, mid, and high dose groups, respectively).
Though considered not biologically significant, a treatment-related  influence on these endpoints could not be excluded.
- Litter size and weights: mean weights 3.64; 3.61; 3.64; 3.56 g = not  affected
- Number viable: Total number of live fetuses 12.4; 13.7; 13.0; 12.6  (control / low / mid / high dose) = not affected. No dead fetuses.
- Sex ratio: 44.4; 46.3; 44.6; 52.4% males (control / low / mid / high  dose) = not affected
- Grossly visible abnormalities: no substance-related findings
- External abnormalities: no substance-related findings
- Soft tissue abnormalities: no substance-related findings
- Skeletal abnormalities: Isolated findings of statistical significance  for high-dosed fetuses at the thoracic vertebra centra and in the 

rib  (here also for low-dosed fetuses): There was 1 fetus (out of 152, i.e.  0.7%) in the high dose group with a tail aplasia and 

spina bifida occulta  as a major defect, associated with several skeletal minor defects on the  vertebra and skeletal retardations. 

This complex finding was associated  secondary to insufficient oxygen supply of this fetus, which is known to  occur incidentally 

during embryonal development within relatively large  litters (14 fetuses in total in this litter). In the absence of  correlating findings 

either in other fetuses or other litters of this  group, these findings were considered to be incidental.
Minor skeletal defects of statistical significance included  aplasia/fused/fragmented thoracic vertrebra centra in 0% (control),  0.6%  (low dose), 0% (mid dose), and 2.0% (high dose, p<=0.05) animals. As the  incidences were only slightly above inhouse 

control data (0-1.5%) and did  not follow a dose response relationship, they were considered to be  incidental. Another minor defect 

of statistical significance was uni- or  bilateral knoddy ribs in 0% (control), 3.2% (low dose, p<=0.05), 0% (mid  dose), and 7.9% 

(high dose, p<=0.05) animals. As historical control data  were not yet available for this endpoint and the occurrence of this  effect 

did not follow a dose response relationship, it was considered to  be incidental.

Applicant's summary and conclusion

Conclusions:
Regarding to the outcome of this developmental toxicity test there is no evidence of developmental toxicity in rats for Tetrahydronaphthalene, according to the classification criteria of Commission Directive 2001/59/EC.
Executive summary:

Daily oral (gavage) administration of Tetrahydronaphthalene at a dose level of 135 mg/kg bw/day during the sensitive phase of organogenesis and intrauterine development (days 6 – 20) of the fetuses induced decreased body weight gains and food intake during the treatment phase, which were interpreted as a first sign of maternal toxicity, without significantly altering pregnancy of the dams and intrauterine development of the conceptuses.

There was no maternal or embryofetal/developmental toxicity observed after administration of Tetrahydronaphthalene at either 45 or 15 mg/kg body weight/day. Tetrahydronaphthalene was not teratogenic in the rat.

The 'No Observed Adverse Effect Level' (NOAEL) was 45 and 135 mg/ kg body weight/day for maternal and developmental effects, respectively.