1,4:5,8-Dimethanonaphthalene, 2-ethylidene-1,2,3,4,4a,5,8,8a-octahydro-

Endpoint summary

Description of key information

NOAEL Oral 28 days, male rats = 75 ppm in diet, equivalent to 7 mg/kg bw/day.  LOAEL Oral 28 days, male rats = 150 ppm (13 mg/kg bw/day).
NOAEL Oral 28 days, female rats = 750 ppm in diet, equivalent to 61 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Mar 1990 - 26 Apr 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted in compliance with GLP; although no official test guideline was named in the study report, the methodology used was essentially consistant with official guidelines (such as OECD TG 407), and so the study is considered reliable with restrictions.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Study methodology was essentially consistent with guideline, but no guideline was recorded in the Study Report.

Deviations:

not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: West- Germany
- Age at study initiation: ca. 4 weeks old
- Weight at study initiation: 131.2 g to 177.3 g (males) and 110.4 g to 144.3 g ( females)
- Housing: The rats were housed in groups of 5, males and females separated, in suspended, stainless steel cages, fitted with wire mesh floor and front.
For each sex the cages were randomly allocated to the various groups.
Each cage was provided with coloured card showing the animal identification numbers, the cage number, the group letter and the study number.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 60-75
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From:15 Mar 1990 To: 12 Apr 1990 (main study) ; the rats of the recovery study were continued without treatment for a period of 14 days and sacrificed on 26 Apr 1990

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Prepared twice during the course of the study.
- Mixing appropriate amounts with (Type of food): Basal diet.
- Storage temperature of food: -20°C

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

Continuous (in diet).

Remarks:

Doses / Concentrations:
0, 150,300 or 1500 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

5
5 additional animals per sex in the control and high dose level groups were used also.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:The dose levels were selected by the sponsor
- Rationale for animal assignment (if not random): random

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (The general condition and behaviour of all animals were checked by cage- site observations)
- Time schedule: twice daily on working days and once a day on Saturdays, Sundays and public holidays (April 13 and 16, 1990)

BODY WEIGHT: Yes
- Time schedule for examinations: initially and at weekly intervals thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Food intake was measured per cage (5 rats/cage) per week by weighing the feeders, during the treatment period and the recovery period. The efficiency of food utilization was calculated and expressed as gram weight gain per gram food consumed

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Water consumption was measured per cage (5 rats/cage)
- Time schedule for examinations: Weekly on 4 consecutive days per week by weighing the water bottles
- This was done during the treatment period only

HAEMATOLOGY:
- Time schedule for collection of blood: on nominal day 28
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: all animals
- Parameters checked : haemoglobin, packed cell volume, red blood cell count, red blood cell distribution width (RDW-SD), total white blood cell count, differential white blood cell count, prothrombin time (was also determined at the end of the recovery period - on nominal day 42), thrombocytes, mean plateled volume (MPV), platelet distribution width (PDW), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC),

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 26
- Animals fasted: No data
- How many animals: all rats
- Parameters checked: alkaline phosphatase activity, alanine aminotransferase activity, aspartate aminotransferase activity, gamma glutamyl transferase activitytotal protein, albumin, ratio albumin to globulin, urea, creatinine, bilirubin (total), sodium, potassium, calcium, chloride, inorganic phosphate, cholesterol, triglycerides. Plasma cholesterol and urea were also determined in the rats killed at the end of the recovery period (on nominal day 42)

URINALYSIS: Yes
- Time schedule for collection of urine: on nominal day 25-26
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: volume, density, appearance, pH, occult blood, protein, glucose, ketone bodies, sediment: erythrocytes, leucocytes, epithelial cells, amorph material, crystals, casts, bacteria, sperm cells, worm eggs. all the urinary parameters, except for volume and density, were also examined in the recovery period, viz. on nominal day 40.

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table below)
HISTOPATHOLOGY: Yes : liver, kidneys, heart, adrenals, and spleen were all examined microscopically.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Prothrombin time was increased in males of the top- dose group at the end of the treatment period but not at the end of the recovery period. The other haematology parameters were unremarkable.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A decreased cholesterol concentration and an increased urea concentration were observed in males of the top-dose group. At the end of recovery period , there were no diffrences other than an increased plasma urea concentration in females.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The relative liver weight of top-dose females and the relative kidney weight of top- dose males were increased at the end of treatment.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Refer below

Histopathological findings: neoplastic:

not specified

Details on results:

Histopathology:
At the end of the administration period there were treatment-related renal changes in males, summarized as proximal tubular degeneration. The proximal tubular cells showed rounded intracytoplasmic hyaline inclusions of varying size, some resembling proteinaceous droplets, others resembling (deeply stained) acidophilic bodies. The renal tubules showed cellular degeneration and desquamation of epithelial cells. These changes occurred in males of all treatment groups, and were most severe at the highest dose level. At this level, kidneys also showed an increased number of basophilic tubules, possibly representing tubular regeneration. No tubular degeneration was observed in females. At the end of the 14-day recovery period all top-dose males still showed proximal tubular degeneration, though much less pronounced than at the end of the 28-day treatment period, and increased numbers of basophilic, most probably regenerating, tubules.

Dose descriptor:

NOAEL

Effect level:

750 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Equivalent to approximately 61 mg/kg bodyweight/day.

Dose descriptor:

LOAEL

Effect level:

150 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: On the basis of observed nephrotoxicity. Equivalent to approximately 13 mg/kg bodyweight/day.

Critical effects observed:

not specified

At the end of the administration period there were treatment-related renal changes in males, summarized as proximal tubular degeneration. The proximal tubular cells showed rounded intracytoplasmatic hyaline inclusions of varying size, some resembling proteinaceous droplets, others resembling (deeply stained) acidophilic bodies.

The renal tubules showed cellular degeneration and desquamation of epithelial cells.

These changes occurred in males of all treatments groups and were most severe at the highest dose level.

Conclusions:

It can be concluded that the no-toxic-effect level for ED-TCD is 750 ppm (equivalent to about 61 mg/kg body weight/day) for female rats, and at least 75 ppm (equivalent to about 7 mg/kg body weight/day) but lower than 150 ppm (equivalent to about 13 mg/kg body weight/day) for male rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

7 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two studies (TNO-CIVO, 1990, study ref V90.247, and TNO-CIVO, 1990, study ref V89.383) were conducted to assess the effects of repeat dose administration of ETD to rats by the oral (dietary) route. The methodology employed by each study was broadly consistent with the modern OECD test guideline 407. Both studies were conducted in compliance with GLP.

Dose groups of five rats per sex per dose were allowed free access to diet to which ETD had been added; dose levels were 150, 300, and 1500 ppm in study reference V90.247, and 75, 750, and 7500 ppm in study reference V89.383; a negative control level (provided with untreated diet) was included in each study. Rats were dosed for 28 days and observed for mortality or other signs of toxicity; animals were sacrificed at the end of the treatment period and a pathological examination performed. In each study, supplementary groups of 5 animals per sex were administered the highest dose level (1500 ppm or 7500 ppm) or untreated diet (negative controls) for 28 days, then observed for 14 days post-administration, to assess recovery following administration.

No animals died in either study at any dose level during the treatment or recovery periods. In each study, signs were seen of liver damage (increased prothrombin time seen in males at the 1500 ppm level, and in both sexes at the 7500 ppm level at the end of the treatment period, and increased liver weights in both sexes at 7500 ppm and at 1500 ppm), however evidence of recovery / reversal of these signs were seen following the 14 -day recovery period for the high-level animals in each study.

There were signs of renal damage in males at doses greater than 75 ppm in each study (signs included: increased kidney weight, the presence of occult blood and epithelial cells in urine, and histopathological renal lesions). As this effect was noted in males only, it was concluded that these effects may have been related to a specific toxic syndrome, alpha-2 microglobulin nephropathy, which may occur following exposure to a number of chemicals, principally hydrocarbons. This syndrome appears to be unique to the male rat (which is consistent with the fact that these renal signs were not observed in the female rats), and on this basis ETD is not considered to be of high concern for kidney damage in humans.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Selected study (CIVO, 1990) continues work conducted in an earlier study (CIVO, 1989). The later study included dose levels intermediate to those used in the 1989 study to better assess the effect threshold. Note that both studies are considered reliable as they were both conducted in compliance with GLP and used methodology broadly consistent with the modern OECD Test Guideline 407.

Justification for classification or non-classification

Apparent toxic effects were seen in the liver of animals treated at high doses, however these effects were seen to recover after the end of treatment. With reference to the CLP Regulation (Regulation (EC) 1272/2008), Annex I section 3.9.2.8.1 the effects seen in the liver in these studies are not considered to justify classification for Specific Target Organ Toxicity (STOT, Repeated dose). In addition, although effects were seen in the kidneys of male rats, as stated above because this effect is only seen in male rats, it is not considered to impact on human health, and so for the purposes of classification, kidneys are not considered to be a target organ.

As no data is available on target organ toxicity following repeated exposure by the inhaled or dermal routes, it cannot be stated conclusively that ETD will not cause repeated dose toxicity, and so STOT (repeated dose) is considered inconclusive for classification.