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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-11-11 to 1998-12-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Not a guideline study. However, study carried out to GLP, and data appears well documented and scientifically reasonable.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In male rats, the systemic toxicity (including to the liver and reproductive organs) was assessed following three weeks dietary exposure to the US or EU versions of the phthalate ester, Santicizer(R) 261.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Two products tested: US Santicizer(R) 261 Plasticizer and European Santicizer(R) 261 Plasticizer
- Substance type: no data
- Physical state: no data
- Analytical purity: US Santicizer(R) 261 Plasticizer >98% pure; European Santicizer(R) 261 Plasticizer 98.75% pure
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Lot/batch No.: US Santicizer(R) 261 Plasticizer Lot No. 83198; and European Santicizer(R) 261 Plasticizer Lot No. 7K3033
- Stability under test conditions: no data
- Storage condition of test material: The high and low diets for both the US and EU Santicizers were stable frozen for at least 36 days
- Other: The two Santicizers may contain different relative concentrations of the same components and/or different components.

Test animals

Species:
rat
Strain:
other: Crl: CD(SD)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC,
- Age at study initiation: Probably about 80 days
- Weight at study initiation: 334-360 g
- Housing: Rats individually housed in solid-bottom polycarbonate cages with stainless steel wire lids with Sani-Chip(R) cage bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: A "one-week quarantine"


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 51.2-62.3
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina certified rodent chow(R)
- Storage temperature of food: "frozen"
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stored frozen feed was analysed on 5 January 1999 [no further details given in study report]
Duration of treatment / exposure:
21 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
75, 750 and 1500 mg/kg bw/day
Basis:
other: target dose levels
Remarks:
Doses / Concentrations:
about 60, 600 and 1200 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
6 males/dose
Control animals:
yes, plain diet
other: positive control
Details on study design:
- Rationale for animal assignment (if not random): by a randomization procedure, stratified by body weight
Positive control:
Di(2-ethylhexyl) phthalate (DEHP), a known testicular and liver toxin, was examined at 1500 mg/kg bw/day only

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: a.m. and p.m.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily
The cage-side observations included changes in skin and fur, eyes, mucous membranes, respiratory system, circulatory system, autonomic and central nervous sytem, somatomotor activity, and behaviour pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: 0, 7, 14 and 21 days


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: No


CLINICAL CHEMISTRY: No


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER: Peroxisomal Acyl-CoA oxidase activity, an indication of hepatic peroxisome proliferation, was measured in the livers of the controls and treated rats at sacrifice.
Sacrifice and pathology:
GROSS PATHOLOGY: Performed, but no details on extent of examination. Organ weights (liver, brain, testis and epididymis) also assessed
HISTOPATHOLOGY: Limited to one testis from each rat in the high-dose group and in the untreated and positive controls
Other examinations:
Acyl-CoA oxidase activity in the liver assessed
Statistics:
Krusal-Wallis one-way analysis of variance by ranks was used to test for differences among dose groups for each test material. Whenever the results of this test were significant (p<0.05), the Mann-Whitney U test was used to make individual comparisons between untreated controls and test material-exposed groups for each test material for that measure (a one-tailed test was used for all parameters except for body weight, organ weight and feed consumption which were examined in a two-tailed test). Jonckheere's test for k independent samples was used to identify significant dose-response trends for each test material. For these data a Chi-square test for independence was used to analyse for differences among treatment groups, and the Cochran-Armitage test for linear trend of proportions was used to determine dose-related linear trends for each test material. When Chi-square revealed significant (p<0.05) differences between groups, then a one- or two-tailed Fisher's exact probability test was used for pair-wise comparisons between each test material-exposed group and the controls.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
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
All rats survived to scheduled sacrifice. Piloerection was observed in one untreated control animal (on day 7), in 2 males given the EU Santicizer(R) 261 at the top dose on day 1 and in one rat on days 2, 5 and 7, in none of the animals exposed to the US Santicizer(R) 261, and in 2 males exposed to DEHP on day 5 and one rat on day 7. In addition, one rat given the high dose EU Santicizer(R) 261 exhibited a sore shoulder on days 14-21 (relevance to treatment unknown).

BODY WEIGHT AND WEIGHT GAIN
No statistically significant differences in body weight or body weight gain were seen in the rats receiving about 60 mg/kg bw/day (of either the EU or US versions) compared to the untreated controls, whereas both test materials produced a statistically significant and dose-related reduction in body weight gain from 600 mg/kg bw/day, particularly during the first week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
For those rats exposed to the EU version, food consumption was significantly reduced (compared to the untreated controls) at the top dose during the first 2 weeks (and returned to normal thereafter) and also during the second week at 600 mg/kg bw/day. With the US version the reduction in food consumption was evident only at the top dose during the first week. Food consumption was unaffected at the lowest dose of about 60 mg/kg bw/day (for either version). Due to the initial decrease in food intake (which was likely due to palatability problems) in the two test groups (and also in those animals receiving DEHP), phthalate intake was less during the first exposure week. Overall, the mean intakes for the US Santicizer(R) 261 were 62.5, 601.5 and 1206 mg/kg bw/day and for the EU Santicizer(R) 261 were 63.3, 593.1 and 1259 mg/kg bw/day, at 0.1, 1, and 2% in the diet, respectively. The overall mean intake of DEHP at 2% in the diet was 1093.9 mg/kg bw/day.

ORGAN WEIGHTS
Compared with untreated controls, both test materials produced statistically significant increases in relative liver weight, this being evident from 600 mg/kg bw/day with the EU version and at 1200 mg/kg bw/day with the US version. Neither compound affected absolute weights of the epididymis, testis or brain. The positive control, DEHP, produced an increase in relative liver weight and a decrease in absolute testis weight.

GROSS PATHOLOGY
There were apparently no gross necropsy findings at sacrifice (extent of examination unclear from study report).

HISTOPATHOLOGY: NON-NEOPLASTIC
At 1200 mg/kg bw/day, minimal testicular degeneration was seen in 1/6 and 4/6 rats given the US and EU versions, respectively. All six of the rats given DEHP exhibited testicular lesions, while there were none in the six untreated controls.

OTHER FINDINGS:
There was a statistically significant increase in acyl-CoA oxidase activity (an indication of peroxisome proliferation) in the livers of rats given the US or EU versions of Santicizer(R) 261 at 600 and 1200 mg/kg bw/day, when compared with untreated controls. The same effect was also observed in the positive controls given DEHP.

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 60 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a 3-week feeding study in which male rats were given two different formulations of Santicizer(R) 261 (one EU and one US) at around 60, 600 and 1200 mg/kg bw/day, effects observed from 600 mg/kg bw/day included reduced body weight gain and increases in liver weight and acyl-CoA oxidase activity (an indication of peroxisome proliferation). The EU version of the test material had a slightly greater effect, but the difference between the two versions was minimal.
Executive summary:

Groups of six male rats were fed either the US or EU versions of Santicizer(R) 261 at 0 (untreated controls), 1 or 2% in the diet, or DEHP at 2% in the diet (positive control), for 3 weeks. Overall, these dietary concentrations corresponded to actual mean intakes for the US test material of 62.5, 601.5 and 1206 mg/kg bw/day and for the EU version of 63.3, 593.1 and 1259 mg/kg bw/day. The overall mean intake of DEHP at 2% in the diet was 1093.9 mg/kg bw/day.

 

There were no deaths during the 3-week exposure period. Piloerection (a non-specific indicator of stress) was observed in one untreated control rat, and in "one to two" males in the high-dose EU Santicizer(R) 261 group and the DEHP group. One rat given the high-dose of the EU version exhibited a sore shoulder in the third week of exposure, but the relevance to treatment is not discussed in the study report.

 

No statistically significant differences in body weight or body weight gain were seen in the rats receiving about 60 mg/kg bw/day (of either the EU or US versions) compared to the untreated controls, whereas both test materials produced a statistically significant and dose-related reduction in body weight gain from 600 mg/kg bw/day (probably related to the significant reduction in food consumption seen at these higher doses).

 

Compared with untreated controls, both test materials produced statistically significant increases in relative liver weight, this being evident from 600 mg/kg bw/day with the EU version and at 1200 mg/kg bw/day with the US version. Neither compound affected absolute weights of the epididymis, testis or brain. The positive control, DEHP, produced an increase in relative liver weight and a decrease in absolute testis weight.

 

There were apparently no gross necropsy findings at sacrifice, although the extent of examination is unclear from the study report. At 1200 mg/kg bw/day, minimal testicular degeneration was seen in 1/6 and 4/6 rats given the US and EU versions, respectively. All six of the rats given DEHP exhibited testicular lesions, while there were none in the six untreated controls.

 

There was a statistically significant increase in acyl-CoA oxidase activity (an indication of peroxisome proliferation) in the livers of rats given the US or EU versions of Santicizer(R) 261 at 600 and 1200 mg/kg bw/day, when compared with untreated controls. The same effect was also observed in the positive controls given DEHP.

 

In conclusion, no statistically significant adverse effects were seen in male rats ingesting about 60 mg/kg bw/day (considered the NOAEL for this study) of either the EU or US versions of Santicizer(R) 261 for 3 weeks, compared to the untreated control animals. At 600 mg/kg bw/day (considered the LOAEL for this study) the male rats consuming the EU version for 3 weeks gained significantly less body weight and had increased liver weights, compared to the untreated controls. In addition at this dose, increased acyl-CoA oxidase activity was seen in the livers of the animals ingesting either the EU or US versions of Santicizer(R) 261.