Ethanol, 2-methoxy-, manufacture of, by-products from, esters with boric acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1992-06-02 to 1993-10-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is well documented and performed according to GLP and generally valid and/or internationally accepted testing guidelines. As the percentage of B-TEGME in the test material is limited, it is not considered as a key study, but as a supporting study for the 90 day study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Brake Fluid DOT 4 (lot 4200) containing B-TTEGME

IUPAC Name:

Brake Fluid DOT 4 (lot 4200) containing B-TTEGME

Details on test material:

- Name of test material (as cited in study report): Brake fluid 500 DOT 4
- Substance type: Borated Glycol Ether
- Physical state: Clear colourless liquid
- Analytical purity: 38% B-TTEGME
- Impurities (identity and concentrations): Confidential details on test material
- Composition of test material, percentage of components: Confidential details on test material
- Purity test date: Not provided
- Lot/batch No.: 4200 (SNC Sample No.); Indent 9450/9486; Toxicology Ref. No. ST91/267
- Expiration date of the lot/batch: Not provided
- Stability under test conditions: Not provided

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Manston, UK
- Age at study initiation: not reported
- Weight at study initiation: not reported
- Housing: single; polypropylene cages (pattern RB3R North Kent Plastics, Dartford);
- Diet: pelleted diet (Maintenance Diet No. 1, Expanded, Special Diets Services), ad libitum
- Water: public supply, ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 45-70
- Air changes (per hr): Air conditioning
- Photoperiod (hrs dark / hrs light): 12/12
- No indications of bacterial contamination (each week a blood agar plate was exposed to air for one hour)

IN-LIFE DATES: From: 1992-06-02 To: 1992-07-17

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Animals were dosed by oral gavage per day. Volumes administered were 0, 0.024, 0.142 and 0.943 ml/kg/day for the control, low, mid and high dose groups, corresponding to the 0, 25, 150 and 1000 mg brake fluid/kg bw/day dosages.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The test material was not analytically verified for the experiment as the material was used with a valid certificate of analysis.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 25, 150 and 1000 mg/kg bw/day
Basis:
other: brake fluid - oral gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 9.5, 57 and 380 mg/kg bw/day
Basis:
other: B-TTEGME - oral gavage

No. of animals per sex per dose:

5 males & 5 females/group

Details on study design:

- Dose selection rationale: Doses were selected based on a range finding study (No. 5249 from Sittingbourne research Centre).
- Rationale for animal assignment (if not random): Animals were allocated to treatment groups using a stratified body weight based computer program (ANALOC). Experimental animal numbers were then randomly allocated to animals.
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the week and once daily on weekends and and holidays for clinical signs.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT & FOOD CONSUMPTION: Yes (See Table 2 for food consumption)
- Time schedule for examinations: Body weight and food intake were measured weekly.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 4 of the study
- Anaesthetic used for blood collection: Yes (CO2/O2 narcosis)
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: white blood cells, red blood cells, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, platelets, red cell distribution width, platelet distribution width, mean platelet volume, platocrit, differential white blood cell count, atypical lymphocytes, normocytes, microcytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 4 of the study
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: sodium, potassium, chloride, protein, albumin, urea, glucose, bilirubin, aspartate aminotransferase, alanine, aminotransferase, alkaline phosphatase, gamma globulin, cholesterol, triglycerides, calcium, inorganic phosphate, creatinine, albumin/globulin ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: during week 4 of the study
- Metabolism cages used for collection of urine: glass metablows
- Animals fasted: Yes (food and water)
- Parameters examined: epithelial cells, tubular epithelium, leucocytes, erythrocytes, sperm cells, casts, bacteria, triphosphate crystals, ammonium phosphate crystals, oxalates, urates, mucus, osmolality, volume, colour, clarity, pH, protein, ketones, bilirubin

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY AND ORGAN WEIGHTS: Yes (see Table 3 for liver weight)
- A detailed post-mortem examination was performed. All gross abnormalities were recorded.
- Organ weights were measured: spleen, heart, brain, kidneys, liver, testes, adrenals, thymus, prostate, epidydimides.

HISTOPATHOLOGY: Yes (see Table 4 for liver changes)
- Histopathology was performed on tissues embedded in paraffin wax and stained with H&E stain.: Adrenals, brain, cervix, epididymides, eyes, heart, intestines, kidneys, liver, nerves (sciatic), ovaries, seminal vesicles, spinal cord, spleen, testes, and thymus.

Other examinations:

Bone marrow smears were prepared from unfixed femur and fixed in methanol.

Statistics:

Two-way ANOVA analysis was performed where suitable. Williams' test was used to test for significance unless a dose-respons was not apparent in which case Dunnett's test was used. Wilcoxon 2 sample rank sum test was also used for haematology, clinical chemistry and urinalysis. Fisher's test (pairwise) was used for histopathology.

Results and discussion

Results of examinations

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):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no treatment related deaths and no treatment related clinical signs.
One male animal (150 mg brake fluid/kg bw/day) was found dead on day 8 without previous clinical signs. The finding of patchy alveolar and bronchiolar oedema suggest that a portion of the dose might have entered the lungs. This death was considered to be due to a handling error.

BODY WEIGHT AND FOOD CONSUMPTION
No adverse effects on body weight.
Food intake was unaltered up to 150 mg brake fluid/kg bw/day. At 1000 mg brake fluid/kg there was a transient and slight but statistically significant decrease (-11%) in food consumption in males for the first two weeks of the study, which then was not apparent for the remainder of the study. No such effect was seen in females. See Table 2.

HAEMATOLOGY, CLINICAL CHEMISTRY andURINALYSIS
No treatment related adverse effects.

ORGAN WEIGHTS and GROSS PATHOLOGY
No treatment related adverse effects.
Liver weights were tabulated below (Table 3).

HISTOPATHOLOGY
No treatment related adverse effects up to 150 mg brake fluid/kg bw/day. The only findings was slight liver centrilobular hypertrophy in five males and three females dosed to 1000 mg brake fluid/kg bw/day (see Table 4). In the absence of any evidence of hepatotoxicity this was considered to reflect physiological adaptation to metabolism of a xenobiotic.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Brake fluid A transient decrease in food consumption, and a very slight centrilobular hypertrophy in 3 females and all males at the 1000 mg/kg dose level, which was not considered to be adverse.

2

Dose descriptor:

NOAEL

Effect level:

>= 380 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: B-TTEGME

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 2. Food consumption (g/animal/week)

Sex	Week	Control	25 mg/kg bw/day	150 mg/kg bw/day	1000 mg/kg bw/day
Males	1	223.8	207.7	219.3	200.2 *
	2	219.1	210.2	212.6	194.1 *
	3	215.9	215.5	217.6	201.5
	4	153.7	149.9	145.7	143.8
Females	1	161.7	156.3	155.9	162.1
	2	157.0	154.0	153.0	152.0
	3	149.2	158.8	149.8	166.8
	4	141.9	144.6	131.2	149.2

* p <= 0.05 significance of difference with control (Williams’s test) Table 3. Mean liver weight at necropsy

Sex	Control	25 mg/kg bw/day	150 mg/kg bw/day	1000 mg/kg bw/day
Males (gram)	11.82	11.70	11.80	12.64
% versus control		99,0	99,8	106,9
Females (gram)	7.52	7.70	7.36	7.73
% versus control		102,4	97,9	102,8

Table 4. Incidences of centrilobular hypertrophy at histopathology

Sex	Control	25 mg/kg bw/day	150 mg/kg bw/day	1000 mg/kg bw/day
Males	0/5	0/5	0/5	5/5 **
Females	0/5	0/5	0/5	3/5

** p < 0.01 significance of difference with control (Fisher’s test)

Applicant's summary and conclusion

Conclusions:

NOAEL: 1000 mg brake fluid/kg bw/day or greater as determined in a 28-day repeated dose toxicity study, corresponding with a NOAEL of 380 mg B-TTEGME/kg bw/day.

Executive summary:

Brake Fluid DOT 4 is considered to have a similar toxicological profile as B-TTEGME. Brake fluid containing 38% B-TTEGME (of which 17% B-TEGME) was administered to groups of five rats/sex at doses of 0, 25, 150 and 1000 mg/kg/day for 28 days, corresponding to 0, 9.5, 57 and 380 mg B-TTEGME/kg bw/day. This did not result in clinical signs, effects on body weight, clinical chemistry, haematology, gross pathology, organ weight and urinalysis changes. The only finding related to treatment histologically was a very slight hepatocellular centrilobular hypertrophy in three females and five males at 1000 mg/kg bw/day. Considering the mild nature of the finding, and in the absence of any other relevant treatment related effect, this was not considered to constitute an adverse effect. The No Observed Adverse Effect Level (NOAEL) was therefore considered to be 380 mg B-TTEGME/kg/day.