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Diss Factsheets

Administrative data

Description of key information

The test substance was determined to have a NOAEL of 400 mg/kg bw/d in propylene glycol in rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984-05 to 1989-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Experimental study conducted similar to OECD guideline and according to GLP
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
The test substance was orally administered to rats for 13 weeks.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Portage, Michigan, USA
- Age at study initiation: 28+/-1 d
- Weight at study initiation: mean body weights of groups were comparable
- Fasting period before study:
- Housing: polypropylene cages with stainless steel grids and lids
- Diet: ad libitum, Labsure CRM pelleted diet (autoclaved)
- Water: ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-2
- Humidity (%): 50+/-10
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
For 7 days before treatment the animals were dosed daily with water to become accustomed to the oral dosing procedure.
The dose volume was 0.5 mL/100 g bw for all animals.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
200, 400 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
16
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: allocation, day before first treatment, day of first treatment and afterwards once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: allocation and afterwards once weekly

FOOD CONSUMPTION:
- Recorded for each cage weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during week before dosing, during week 13
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: timepoint 1: week 7, timepoint 2: after dosing period (from cardiac puncture)
- Anaesthetic used for blood collection: timepoint 1+2: No data
- Animals fasted: timepoint 1+2: No data
- How many animals: timepoint 1: 8 of each sex from each group, timepoint 2: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: timepoint 1: week 7, timepoint 2: after dosing period (from cardiac puncture)
- Animals fasted: timepoint 1+2: No data
- How many animals: timepoint 1: 8 of each sex from each group, timepoint 2: all animals

URINALYSIS: Yes
- Time schedule for collection of urine: week 7 and 13
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, partially. First collection overnight (16 h) and subsequent 6 h period without water

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Femoral bone marrow samples were taken from all rats and smears prepared for examination after the dosing period.
Sacrifice and pathology:
Animals were sacrificied after 1, 2, 4, 6 or 24 h after final dose. The cardiac blood samples were collected afterwards.
GROSS PATHOLOGY: Yes, organs weighted: adrenals, brain, heart, kidneys, liver, pituitary, spleen, thyroids (with parathyroids) and, either testes, prostate with seminal vesicles, or ovaries.
HISTOPATHOLOGY: Yes, samples taken from adrenals, aorta, bone (femur), brain, caecum, cochleae, colon, duodenum, eyes, heart, ileum, kidneys, liver, lungs (after inflation with fixative), lymph nodes (cervical, mesenteric and inguinal), mammary gland, oesophagus, pancreas, pituitary, salivary gland (submaxillary), sciaric nerve, skeletal muscle (biceps femoris), skin, spinal cord, spleen, stomach (secretory and forestomach), thyroids (with parathyroids), trachea, thymus, tongue, urinary bladder, and either: testes and prostate with seminal vesicles or ovaries and uterus with cervix and vagina.
All the organs and tissues listed above, with the exception of cochlea, were examined from all control rats and all rats given 400 mg/kg bw/d. In addition, sections of the stomach and liver were examined from all rats given 200 mg/kg bw/d. The right cochlea from five male and five female rats in each of the control and high-dose groups were examined by light microscopy.
Other examinations:
One day after arrival, five rats of each sex were killed and a comprehensive microbiological examination was conducted. A similar examination was carried out on five male and five female Sprague-Dawley rats obtained from the Clinical Research Centre (CRC). A further five male and five female CRC rats were housed in the same room as the study, as sentinels, under identical conditions. These rats were killed during week 14 and a comprehensive microbiological examination conducted. The findings of the examination on the Charles River CD rats indicated that their health status was good, and that they were of a suitable high quality for the study. Results of the examinations of the CRC rats indicated that they had not been exposed to any subclinical infections during the course of the study.
Statistics:
The methods used are as described by Chappell and Scott. Significance testing was performed at p < 0.05 and p < 0.01.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Two deaths occured. One animal from 400 mg/kg bw treatment group on day 3 and one animal from 200 mg/kg bw/d treatment group on day 80. Reasons for death were not determined.
Increased salvation and stained coats were observed in all groups.
Rales and uneven breathing were observed occasionally in 12 males and 13 females of 400 mg/kg bw/d group and for 2 males and 4 females in 200 mg/kg bw/d group. One controll animal was once observed with rales.
2 females of 400 mg/kg bw /dgroup collapsed after treatment on day 81 but recovered during the same day.

BODY WEIGHT AND WEIGHT GAIN
Slightly lower bodyweight gains were recorded for treated females in comparison to the controls, though the differences were not statistically significant and not dose dependent. The weight gains of males were not affected by treatment.

FOOD CONSUMPTION
Slightly higher mean weekly food consumption was recorded throughout the dosing period for treated males in comparison to the controls. Food consumption of female rats was not affected by treatment.

OPHTHALMOSCOPIC EXAMINATION
No effects detected.

HAEMATOLOGY
There was no clear effect of treatment on haematological parameters. At week 7, treated females had slightly increased erythrocyte size, as evidenced by high PCV and MCV values, and low MCHC. These differences were greater for females given 200 mg/kg bw/d than those given 400 mg/kg bw/d. Furthermore, erythrocyte size was normal in treated males at week 7, and normal in both sexes after 13 weeks' treatment. Consequently, the difference at week 7 is considered to be of doubtful significance. There were no other differences between treated and control rats. There was no effect of treatment on myelograms.

CLINICAL CHEMISTRY
Higher alkaline phosphatase (ALB) and cholesterol values were recorded for treated males after 6 and 13 weeks of treatment in all dose groups. A trend towards higher ALB values was also noted for treated females, though the differences from controls were not statistically significant. Treated males had higher serum urea and creatinine levels at week 7, but not after 13 weeks' treatment. Lower gamma globulin levels were recorded for males and females given 400 mg/kg bw/d at both investigations, and for males and females given 200 mg/kg bw/d after 13 weeks of treatment. Minor changes in other protein fractions were recorded for females at the higher dose. These small changes were not consistent and were not considered to be a response to treatment. At the terminal investigations, blood samples were collected over two days. Urea, triglyceride and potassium values were affected by the day of sampling. However, there was no difference between treated and control rats an each occasion.

URINALYSIS
Treatment was associated with slightly lower urinary pH in males, and there was a tendency towards higher urinary flow rate for rats given 400 mg/kg bw/d. Examination of the urinary sediment revealed increased epithelial cell counts for treated males at week 7, and of cellular detritus in the samples of treated males at both investigations. Males given 200 mg/kg bw/d showed poor concentrating ability at both collections but, since this appeared to result from high initial specific gravity in rats given 200 mg/kg bw/d and was not apparent at the higher dosage, it was considered not to be due to treatment.

ORGAN WEIGHTS
Liver and kidney weights were elevated in treated males, and liver weights were elevated in females given 400 mg/kg bw/d. Statistical analysis also revealed significantly lower pituitary weights for treated females. Since the differences were not dosage-related and all individual values were normal, this was considered to be of no biological significance.

GROSS PATHOLOGY
Treatment-related changes were found in the stomachs. The changes comprised discrete or diffuse raised areas on the mucosal surface (particularly in the forestomach), a wrinkled, rough or discoloured appearance of the forestomach mucosa, thickening of the limiting ridge and prominence of the stomach blood vessels. The most marked change was the presence of raised areas, and this was recorded for all males given 400 mg/kg bw and killed after 13 weeks treatment, for six females given this dose, and for one female given 200 mg/kg bw/d. Other changes were seen in all but one of the remaining females given 400 mg/kg bw/d and in 11 males and twelve females given 200 mg/kg bw/d. Discolorations of the mucosa of the secretory region of the stomach, either generally or as discrete foci or areas, were also recorded in a few treated rats. There was no macroscopic finding in other tissues that was considered to be related to treatment.

HISTOPATHOLOGY:
In the liver, centrilobular hepatocyte enlargement or centrilobular glycogen loss were recorded for most rats given 400 mg/kg bw/d. These changes were not present in rats given 200 mg/kg bw/d or in control rats.
In the stomach, treatment-related changes were seen in the forestomach. There were no changes in the secretory stomach. The lesions comprised epithelial damage and submucosal oedema, thickening of the epithelium, and changes in the keratin layer. Epithelial damage, that is ulceration, erosion or necrosis, was seen in one male and five females given 400 mg/kg bw/d. Submucosal oedema was seen in several rats given 400 mg/kg bw/d. In the females it was associated with epithelial damage. Thickening of the forestomach epithelium, apparent as plaques or diffuse areas, and resulting from hyperplasia, was seen in rats given 400 mg/kg bw/d. This hyperplasic change was not seen in rats given the vehicle or 200 mg/kg bw/d. Minimal generalized thickening of the forestomach epithelium was apparent in one rat given 400 mg/kg bw, nine given 200 mg/kg bw and one propylene glycol control. The higher incidence of this feature in the 200 mg/kg bw/d group may have been related to treatment. Changes in the keratin of the forestomach comprised hyperkeratosis, swelling of the keratin layers and, in one female given 400 mg/kg bw/d, a marked hydropic change. Hyperkeratosis, was recorded for most rats given 400 mg/kg bw/d, for two males given 200 mg/kg bw/d and for one control male. The distinctive swelling of the keratin, often associated with separation of the keratin layers, was seen in most treated rats but was not present in the controls.
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Dose descriptor:
NOAEL
Effect level:
< 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local effects in forestomach
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The experimental data are considered reliable and further supported with other studies. The relevance of observed forestomach lesions for human is limited (please refer to Discussion).

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

Oral

Key study

The test substance was examined for its subchronic toxicity in a 13 week repeated oral dose study, similar to OECD 408 and according to GLP (BASFSE.1989, TX89028). Groups of 16 male and 16 female Charles River CD rats were administered the test substance in propylene glycol at dose levels of 0 (control group), 200 and 400 mg/kg bw/d. The test substance was administered by oral gavage daily for 13 weeks. Signs of toxicity were recorded continuously. Body weights and food consumption were recorded weekly. Hematology, blood biochemistry and urinalysis were conducted after 6 and 13 weeks. After 13 weeks of treatment, all animals were sacrificed, dissected and examined macroscopically and microscopically. Body weight gain was marginally decreased in the treated females only but showing no clear dose-relationship.. Food consumption was slightly increased in males at 400 mg/kg bw/d only but this was considered not as an adverse observation. Hematology and ophthalmoscopy revealed no substance-related changes. Administration of the test substance produced predominantly changes in the forestomach epithelium. In the high dose group, frank epithelial damage, thickening of the epithelium due to hyperplasia, and hyperkeratinosis were detected in all males and 6/16 females. According to the authors, such changes were indicative of a reactive response to an irritant. Generalized and minimal epithelial thickening of the forestomach were also observed in some low dose rats and in one control rat. However, the tissue was not hyperplastic. A distinctive swelling of the keratin layer in the forestomach epithelium was also observed at both dose levels. In addition, increased salivation following dosing was noted in the treated rats. According to the authors, this may also be indicators of a response to an irritant. Increased serum ALP activities with statistically significance were noted intermittently only in males, while no statistically significant deviations were noted at the end of treatment in both sexes and both doses. Further, substance-related functional changes were recorded for the liver and kidneys. Increased liver weights and increased cholesterol levels were found in both treated groups, but mild histological changes (centrilobular hepatocyte enlargement and glycogen depletion) indicative for a slightly impaired liver function rather than a clear toxic effect were limited to the high dose group. Functional renal changes comprised of increases in kidney weight, urine flow, urinary acidity, urine epithelial cell content, and serum urea and creatinine. However, as there were no histopathological changes associated with these functional alterations, these observations were also considered as not adverse in nature. Finally, as no clear or adverse substance related signs of systemic toxicity were observed, the systemic NOAEL was considered to be 400 mg/kg bw/d in male and female rats. With regards to the local, irritative effects at the forestomach as port of entry, the local NOAEL was considered to be < 200 mg/kg bw/d.

 

Supporting studies

An explorative repeated dose toxicity study was conducted in rats via the oral route (BASF SE.1989, TX89029). Only 56 male animals were treated for up to 13 weeks with 400 mg/kg bw/d of the test substance via gavage. Control animals (56, male) received the vehicle alone. The study evaluated the onset and progress of pathological changes observed in the stomachs of rats treated with the test substance. Six or 8 rats per group were sacrificed after 1, 3, 5, 7, 9, 11, and 13 weeks of dosing. All animals were observed for clinical signs of toxicity, data on body weights and food consumption were recorded. After sacrifice, the stomach of each rat was removed and examined macroscopically and microscopically. No deaths occurred. Body weight gain of the treated rats was lower than control. In the treated group, increased salivation and increased incidence of body stains, damp hair, respiratory problems and nasal exudate (possibly associated with the increased salivation) was observed. Macroscopic examination revealed treatment-related changes of the mucosal surface and the limiting ridge of the forestomach. Raised areas in the forestomach were noted first after 1 week of dosing and were seen in all treated rats after 5 weeks. A more generalized thickening or wrinkling of the limiting ridge and mucosa was observed after 5 and 7 weeks, respectively. Microscopically, the changes in the forestomach resembled a reactive response to an irritant. Distinctive swelling of the keratin layer was seen in most of the treated rats after 1 week of dosing and in all treated rats after 3 weeks. Epithelial hyperplasia was observed in one rat after 1 week and in all treated rats from week 7 onwards. Hyperkeratinosis at the limiting ridge was observed first at 5 weeks and was present in all treated rats from week 11 onwards. Ulceration of the epithelium of the forestomach was seen in a few rats. There were no treatment-related findings in the secretory region of the stomach. These results showed that hyperplasia of the forestomach epithelium was established in all rats after 7 weeks of dosing. Hyperkeratinosis and ulceration were also present at that time. The severity of these lesions was unaltered by further 6 weeks of dosing.

 

A further explorative 7 week repeated dose study was conducted to investigate lesions of the forestomach of male rats induced by the test substance with regard to reversibility and vehicle effects and to establish a no-effect-level (BASF SE.1989, TX89030). The test substance was administered by gavage to male Charles River Sprague-Dawley CD rats at a dose of 400 mg/kg bw/d using two different vehicles. After 7 weeks of dosing, some of the rats were observed for 4 or 8 weeks while others were sacrificed directly. All animals were observed for clinical signs of toxicity. Data on body weights and food consumption were recorded. After sacrifice, the stomach was removed and examined macroscopically and microscopically. Only half of the animals sacrificed after 8 weeks of recovery were examined. One rat died on day 2 and one on day 24 of treatment. Both deaths were established to be due to intubation errors. Increased salivation was observed in most of the rats given the test substance and in a few control rats given propylene glycol. Further clinical signs consisted of an increased incidence of body stains and respiratory problems and may be associated with the increased salivation. Lower body weights were observed in the two groups given 400 mg/kg bw/d. This was reversible in the rats given the test substance in propylene glycol. Treatment-related changes of the mucosal surface and limiting ridge of the forestomach were observed in high dose rats sacrificed immediately after the dosing period. The lesions were more pronounced in rats administered the test substance in propylene glycol. No such lesions were observed in the control, low dose and mid dose groups and in high dose rats sacrificed after 4 or 8 weeks of recovery. Microscopical examination revealed hyperplasia and hyperkeratinosis of the forestomach epithelium in high dose rats sacrificed without recovery, the rats receiving the test substance in propylene glycol being more affected. Ulceration of the forestomach was seen in rats given 400 mg/kg bw/d in propylene glycol. Hyperplasia, ulceration or swollen keratin were not observed in controls, low and mid dose groups, although minimal epithelial thickening and hyperkeratinosis was found in mid dose rats an in one propylene glycol control rat. All lesions were reversible within 8 weeks of recovery. These results suggested that propylene glycol alone produced minor changes to the forestomach epithelium and enhanced the irritant action of the test substance. The findings in rats given 100 mg/kg bw/d in propylene glycol were equivalent to those found in rats given propylene glycol alone. Thus a dose of 100 mg/kg bw/d represented the no-effect-level for forestomach lesions as indication of local irritation using propylene glycol as a vehicle.

 

 

Conclusion

The results of the key study showed no clear or adverse substance related signs of systemic toxicity. The systemic NOAEL was considered to be 400 mg/kg bw/d in male and female rats and with regards to local, irritative effects at the forestomach, the local NOAEL was considered to be < 200 mg/kg bw/d. With the supporting studies the irritative effects caused by the test substance within the forestomach of the rats in the key study was further evaluated. The two studies investigate the onset time, development and reversibility of the pathologic effects. Those studies revealed that severity of lesions remained unchanged after 6 weeks of dosing even after further treatment and were reversible within 4 weeks after dosing for 7 weeks. No further refinement of the NOAELs was derived from the supporting studies. The authors of the studies and a summary report, based on TX89028, TX89029 and TX89030, concluded that the effects of the test substance were typically related to irritant properties of the test substance. The summary report furthermore discussed the relevance of the results from studies conducted in rats to human. After consideration of the anatomical and physiological differences between man and rat, and the reversibility in rats there are very clear indications that the forestomach lesions seen in rats have no relevance to human. Therefore a systemic NOAEL was determined to be 400 mg/kg bw/d in male and female rats which was used for systemic DNEL derivation. The observed local effects on the forestomach were considered for a local NOAEL of < 200 mg/kg bw/d in rats. As the local irritation at the rat’s forestomach has no relevance for human, the local NOAEL was not considered for derivation of local DNELs.

 

Additional studies were conducted with administration of the test substance in a medical preparation. Due to the effects not being clearly substance related and the lack of guideline conformity, the studies were disregarded.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Experimental study conducted similar to OECD guideline 408 and according to GLP.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: stomach

Justification for classification or non-classification

Repeated dose oral toxicity

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data in relation with the expected relevance and application dosage in human are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008, as amended for the seventh time in Regulation (EU) No 2015/1221. As a result the substance is considered to be not classified for repeated dose oral toxicity.