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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Remarks:
US GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): diethylene glycol monobutyl ether
- Physical state: liquid
- Analytical purity: 99.2%
- Other: supplied by Dow Chemical Company, Plaquemines, LA

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: 5 weeks
- Acclimation period: yes


ENVIRONMENTAL CONDITIONS
- no data other than adequate temperature, humidity and photocycle

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Prepared weekly, serial dilution for lower dose levels. Concentration changed weekly to accomodate animal growth. Solutions found to be stable for at least 9 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solutions were between 98.3-102% of target. Test material intake calculated from drinking water consumption to be between 100.7 and 104.8% of target levels for all doses over study period.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Continuous via drinking water.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
target dose
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
target dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
target dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on two week range finder study.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-study and during last week of treatment

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to necropsy from orbital sinus.
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: Yes
- Parameters checked: Hct, Hgb, RBC count, WBC count, platelet count, differential WBC count, RBC indices and morphology, reticulocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to necropsy
- Animals fasted: Yes
- Parameters checked: ALP, ALT, AST, serum albumin, chlolesterol, creatinine, glucose, total bilirubin, total protein, BUN, electrolytes (Ca, Cl, PO4, K, Na)

URINALYSIS: Yes
- Time schedule for collection of urine: overnight during week prior to necropsy
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: No
- Parameters checked: colour, appearance, sg, volume, pH, bilirubin, glucose, protein, ketones, blood, urobilinogen. Urine sediment (pooled per sex/dose) also examined microscopically.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pre-study and during last week of treatment
- Battery of functions tested: sensory evaluation (nociception test, startle response), grip strength, motor activity, rectaul temperature.+
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes. All organs from high dose and control animals plus a selected number from low and mid dose animals. Rats fasted over night then anesthetised with CO2 and complete necropsy on all animals. Tissues examined: brain, liver, kidneys, heart, spleen, adrenals, testes, epididymides, uterus, ovaries, thymus. Bone marrow smears from femurs of all animals. Histological analysis of high and control animals of tissues examined; also lungs, liver, kidneys and spleen (females only) and relevant gross lesions from the middle and low dose groups processed and examined.
Other examinations:
For control and high dose animals, total spermatid count per testis, total sperm counts per epididymidis, total counts per gram of tissue, sperm motility. 200 sperm per animal assessed for morphology. Liver metabolic enzymes from high dose and control animals to assess the levels of mixed function oxidases (CYP1A1, CYP1A2, CYP2B1/2, CYP2E1) and microsomal UGT. Positive controls were used in enzyme assays.
Statistics:
Means and standard deviations for continuous data. All parameters examined tested for equality of variance (Bartlett's test, alpha=0.01) and if significant, transformed to obtain equality of variance. Alpha levels set at 0.05 for interaction between designated variables for dose related effects. Alpha levels also set at 0.05 for interaction terms (with Bonferroni's correction.)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slight reduction in high dose group of both parameters observed over whole study period (10% males, 6% females). All within historical control ranges.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Reduced consumption (7-8%) in high dose group, particularly in males
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No significant effects seen in the range finder study up to 2000mg/kgbw/day. In the main study, RBC and Hgb slightly but statistically significantly reduced in mid and high dose animals (both sexes- 3-8%). Hct similarly slightly reduced in high dose groups. Changes showed a dose response relationship, particularly for males. Results for mid dose group all within laboratory historical control values for dietary studies (male RBC value, which was within historical control for dietary and drinking water studies.) Note however that the study control values were higher than (outside) historical control ranges. (See table below) Also note that no changes to RBC indices or morphology were observed. A small but significant sex-by-dose interaction with reticulocyte was attributed to random variation and not treatment. There were no effects on WBC parameters. The changes in the mid dose group are of questionable biological relevance
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Slight but statistically significant decreases in serum levels of AST, total protein and cholesterol were observed in both sexes at the highest dose.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Consistent with the decreased water consumption, urine specific gravity was increased in the high dose group. Urine pH also decreased with dose but this was attributed to excretion of the metabolite 2-(2-butoxyethoxy)acetic acid.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver (relative) and kidney (relative and absolute) weights increased in the high dose group. Spleen weight increases were observed for the mid and low dose animals but not the high dose group. As these changes were not dose dependent and were within the historical control ranges for dietary studies (if not drinking water studies) they were not regarded as biologically relevant changes (see tables below). No other treatment related and dose dependent organ weight changes were seen in the mid and high dose groups. No changes were seen in other organs.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The only finding attributed to treatment was in female livers in the high dose groups where slight or very slight lesions were recorded (foci of aggregates of macrophages/histiocytes which are common in F344 rats and were observed in greater numbers in the high dose females.) Very slight hypertrophy of periportal hepatocytes was also seen in 6 high dose females. A slightly greater incidence of renal cortical tubule degeneration was also seen (very slight in nature) was seen in the high dose animals. Since this is routinely found at low incidence in F344 rats of this age, the observation could not be regarded as definitive. No treatment related histopathological effects were found in the bone marrow or spleen.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sperm analysis: No effects seen.
Details on results:
Liver enzyme analysis: Activities of all mixed function oxidases generally slightly increased. As only the top dose was examined, it is not possible to relate this to the NOAEL.

Effect levels

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
water consumption and compound intake
haematology
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Changes seen in Erythron values compared to historical control values for 13 week study males

Dose

RBC (E6/µL)

HGB (g/dL)

HCT (%)

Control

9.27 (0.35)

15.8 (0.5)

46.2 (2.4)

50mg/kgbw/day

9.13 (0.22)

15.7 (0.4)

45.5 (1.3)

250mg/kgbw/day

8.94 (0.34)

15.3 (0.4)

44.5 (1.9)

1000mg/kgbw/day

8.53 (0.31)

14.8 (0.4)

42.6 (2.0)

Historic control range in drinking water studies

9.11 – 9.22

15.0 – 15.6

42.6 – 45.2

Historical control range in dietary studies

8.85 – 9.68

15.3 – 16.5

39.8 – 42.7

SD values shown in brackets

 

Changes seen in Erythron values compared to historical control values for 13 week study females

Dose

RBC (E6/µL)

HGB (g/dL)

HCT (%)

Control

8.26 (0.22)

15.6 (0.13)

43.3 (1.1)

50mg/kgbw/day

8.06 (0.31)

15.2 (0.4)

42.3 (1.8)

250mg/kgbw/day

8.07 (0.24)

15.2 (0.4)

42.5 (1.7)

1000mg/kgbw/day

7.54 (0.17)

14.8 (0.3)

40.5 (1.0)

Historic control range in drinking water studies

7.81 – 8.20

15.3 – 16.5

43.6 – 46.8

Historical control range in dietary studies

7.89 – 8.42

14.6 – 15.8

40.6 – 43.6

SD values shown in brackets

Changes seen in Spleen weight values compared to historical control values for 13 week study males

Dose

Absolute (mg)

Relative (mg/100g)

Control

594 (35)

190 (7)

50mg/kgbw/day

638 (21)

198 (8)

250mg/kgbw/day

635 (24)

200 (9)

1000mg/kgbw/day

632 (22)

210 (15)

Historic control range in drinking water studies

567 - 605

185 – 193

Historical control range in dietary studies

536 - 668

175 - 206

SD values shown in brackets

 

Changes seen in Spleen weight values compared to historical control values for 13 week study females

Dose

Absolute (mg)

Relative (mg/100g)

Control

440 (29)

248 (19)

50mg/kgbw/day

448 (35)

255 (18)

250mg/kgbw/day

457 (35)

261 (22)

1000mg/kgbw/day

444 (32)

260 (22)

Historic control range in drinking water studies

401 - 450

242 – 252

Historical control range in dietary studies

380 - 444

226 - 248

SD values shown in brackets

Applicant's summary and conclusion

Conclusions:
The small changes in haematological parameters seen in the mid dose group were within historical control ranges and since they were close to the concurrent control values (within 3%) and these themselves were unusually high, changes were not deemed to be an adverse effect. Changes seen in spleen weight did not show a dose response and were within historical control ranges.
Executive summary:

In a 90 day sub-chronic guideline and GLP drinking water study, standard toxicological end points, supplemented by additional examinations, were studied for Fischer 344 rats in doses up to 1000mg/kg. Multiple, albeit mild, effects were seen in the high dose group. The only treatment related effect seen in the mid dose group were equivocal changes (decreases of around 2 -3%) in erythron (RBC count, Hgb and Hct) that were statistically significant to unusually high concurrent controls but within historical control ranges. Changes in spleen weight did not show a dose reponse and were also within historical control values. This dose level of 250mg/kg/day was therefore considered to be the no adverse effect level.