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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1992-06-02 to 1992-09-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction. The study was conducted according to Good Laboratory Practices and adhered to OECD guideline 408.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Test articles were stored at room temperature, in the dark, and flushed with a stream of nitrogen prior to storage following each occasion of use.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley Inc. (Indianapolis, Indiana)
- Age at study initiation:
- Weight at study initiation:
- Fasting period before study: not reported
- Housing: polypropylene cages with stainless-steel grid tops and floors
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 24°C
- Humidity (%): 45 to 70%
- Air changes (per hr): minimum 15 per hour with no recirculation using high efficiency filters
- Photoperiod (hrs dark / hrs light): 12 hours light/dark


IN-LIFE DATES: From: 1992-06-02 To: 1992-09-01

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): mixed with powdered diet
- Storage temperature of food: room temperature in sealed metal containers
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of diet (between 2 and 10 g, depending on dose level) were extracted on a multiple vortex mixer for 5 minutes and then in an ultrasonic bath at 40°C for 30 minutes with carbon tetrachloride (50 or 100 millilitres). Extraction columns were prepared by weighing 3 grams deactivated Florisil into sintered glass filter tubes (one for each extract to be processed). Deactivated Florisil was prepared by overnight drying at 110°C followed by addition of 0.05 ml water/gram and mixing for 1 hour prior to use. An aliquot of the organic phase from the diet extract was poured onto the extraction column, the eluate collected and the column washed with carbon tetrachloride to a known fmal volume. The infra-red absorbance, in the CH stretching region, of the eluate was measured against a carbon tetrachloride background using a Fourier Transform infra-red spectrometer. The concentration of test article was determined by comparison with standard solutions of the test article.
Duration of treatment / exposure:
90 days continuous exposure through feed
Frequency of treatment:
continuous, ad libitum
Doses / concentrations
Remarks:
Doses / Concentrations:
0.02%, 0.2%, and 2% for paraffin wax 64 and micro/paraffin wax mixture, 2% for low melting point wax
Basis:

No. of animals per sex per dose:
20/sex/dose
Control animals:
yes, plain diet
Details on study design:
Ten-fold decremental dietary concentrations of mineral waxes, from 2% to 0.02% (w/w), were selected based on concentrations where effects were
observed in previous studies. Animals were randomised to their respective treatment groups.
Positive control:
No data provided.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weighed three days before treatment, on first day of treatment, and twice weekly until killed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: examined during the week before treatment and between 4 and 11 days before the end of treatment; examination included the lids, conjunctivae, cornea, anterior chamber, iris, lens, vitreous body and fundus
- Dose groups that were examined: high dose and control animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: collected at necropsy
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: collected at necropsy
- Animals fasted: No data
- How many animals: all animals
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
No data reported.
Statistics:
Analysis of data was performed using the following general procedures as appropriate. The continuous variable data from the control and test groups were tested for normality using the Kolmogorov-Smirnov (K.S.) test and homogeneity of variance using Bartlett's test. Statistical significance was determined to be at p <0.05 in a K.S. test and at p <0.01 in a Bartlett's test. If both tests were non-significant, the control and test groups were compared using analysis of variance followed by the least significant difference (L.S.D.) test. If either test produced a significant result, a suitable transformation was attempted. If the transformed data resulted in a non-significant Bartlett's test but a significant K.S. test, the Wilcoxon Mann-Whitney test was used. If the transformed data resulted in a non-significant K.S. test but a significant Bartlett's test, an appropriate t-test was used, based on whether a pooled variance was suitable or not. If no suitable transformation could be made, one of the above tests was selected as the most appropriate based upon the nature and distribution of the data. Where levels of significance are reported in the tables for transformed data, the means and standard deviations are reported for the untransformed data. In all test comparisons, a probability level of p<0.05 in a two-sided test was taken to indicate statistical significance. Data from the histopathological examination was tested for an increase in incidence between treated and control animals using Fisher's exact test. A probability level of P <0.05 in a one-sided test was taken to indicate statistical significance.

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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
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:
not examined
Details on results:
Data tables with specific values for haematology, body weights, organ weights, and other measurement were not provided in the study report, and therefore only qualitative comparisons are reported in this record.

CLINICAL SIGNS AND MORTALITY
One female animal in the 2.0% micro/paraffin wax mixture group from the reversal study was killed on day 31 of treatment, but this condition was not considered to be treatment-related. No other mortalities or clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN
No treatment-related differences were observed.

FOOD CONSUMPTION AND COMPOUND INTAKE
No treatment-related differences were observed. There was little evidence of any difference in the food intakes between the various groups of either sex during the reversal period of the study.

OPHTHALMOSCOPIC EXAMINATION
No abnormalities existed before treatment, and occasional abnormalities observed after treatment were not considered treatment-related.

HAEMATOLOGY
A reduction in the haemoglobin concentration (Hb) was seen for both males and females treated with 2.0% low melting point wax. A slight increase in
reticulocytes was also seen in these groups. In the females the red blood cell count (RBC) and haematocrit (HCT) were slightly reduced. In the male
2.0% micro/paraffin wax mix group the Hb was reduced, but not as significantly as that seen with 2.0% low melting point wax. No reductions in Hb were seen in the females treated with micro/paraffin wax mix or paraffm wax 64, although an increase in reticulocytes similar to that seen with low melting point wax did occur in the females treated with paraffin wax 64 at a level of 2.0%. In all cases there was no effect on the mean cell volume (MCV)of treated animals. In the males treated with low melting point wax the reductions in Hb resulted in a reduced mean cell haemolobin (MCH) and mean cell haemoglobin concentration (MCHC). Similarly there was a reduction in the MCH of the male 2% micro/paraffm wax mix group. In the reversal study a very small reduction in the MCH of the male group treated with paraffin wax 64 and low melting point wax at a dose level of 2.0% was seen. Otherwise the male animals on the reversal study had normal red cell parameters. For the females only the animals in the 2.0% paraffin wax 64 group showed changes in red cell parameters after the reversal period These involved small reductions in Hb, MCV and MCH with an increased reticulocyte count. In both the male and females treated with 2.0% low melting point wax there was a reduction in the number of platelets. Similar reduction.were also seen at the 2.0% level of micro paraffin wax mix and paraffin wax 64 for both the males and females. The platelet numbers were also reduced in animals treated with 0.2% of both waxes tested at that concentration. An increase in neutrophils was apparent in the animals treated with 2.0% low melting point wax. The increase in neutrophils for females was of greater significance than that of the males. No effect was seen on the prothrombin time in the females. In the males a slight reduction in prothrombin time was seen in the 2.0% micro/paraffin wax mix group and the 2.0% low melting point wax group.

CLINICAL CHEMISTRY
The serum levels of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) were higher than controls in one or more of the groups for each test article in male and female animals. These parameters were also found to be higher than controls at the end of the reversal period for male and female animals treated with micro/paraffin wax mixture or low melting point wax. Alkaline phosphatase (ALKP) was lower than control in male rats treated with 0~2% or 2.0% paraffin wax 64 or micro/paraffin wax mixture at each of the dietary levels tested. This parameter was also reduced,
compared to controls, in the female groups treated with 2.0% paraffin wax 64 or low melting point wax. At the end of the reversal study changes in serum levels of ALKP were only seen in animals treated with low melting point wax. Changes in albumin (ALB) and albumin/globulin ratio (ALB/GLOB) were seen for male and female animals treated with 2.0% micro/paraffin wax mixture. For the males similar changes were also seen in the low melting point wax group. Female animals treated with 2.0% paraffin wax 64 or 0.2% micro/paraffin wax mixture had levels ofALB which were lower than controls. At the end of the reversal period there were no changes in the ALB or ALB/GLOB ratio of male treated animals compared to controls. In the females changes were seen in the ALB levels of the paraffin wax 64 and low melting point was groups. In the low melting point wax .group the ALB/GLOB ratio was also lower than that of the controls. Gamma-glutamyl transferase (gamma-GT) values were higher than controls for female animals treated with 2.0% micro/paraffm wax mixture or low melting point wax. This finding was also observed in these groups and in the female paraffm wax 64 group at the end of the reversal period.

ORGAN WEIGHTS
The mean liver weights of male rats given 2.0% micro/paraffm wax mixture or low melting point wax were higher than control. When liver weight was expressed relative to body weight, higher values than controls were present in both the above groups plus the groups given either 2.0% or 0.2% paraffin wax 64. For female rats all treatment groups receiving 2.0% or 0.2% test article showed both higher absolute and relative liver weights than controls. Animals in the reversal groups generally had liver weights close to control values. However, relative liver weight was still statistically significantly higher than control at the end of the reversal period in male rats receiving 2.0% paraffin wax 64 or low melting point wax and in female rats receiving 2.0% micro/paraffm mixture or low melting point wax.

In the males the absolute and relative weight of the lymph nodes was higher than controls at all levels of treatment with paraffm wax 64, at 0.2% and 2.0% micro/paraffin mixture and in the low melting point wax group. The magnitude of this effect was greatest in the group treated with low melting point wax. Although an effect was seen at a lower level of paraffin wax 64 than of micro/paraffin mixture the magnitude of the effect was greater with 2.0% micro/paraffin wax mixture than with 2.0% paraffin wax 64. Similar differences in the weight of lymph nodes were seen in the female animals. For animals in the reversal groups generally the lymph node weights were still higher than control at the end of the reversal period in the male and female groups previously treated with each of the mineral waxes. The magnitude of the difference was less than that seen immediately after the end of treatment.

Absolute and relative spleen weights were higher than control in both sexes treated with 2.0% micro/paraffin wax mixture or low melting point wax and in females fed diet containing either 0.2% or 2.0% paraffin wax 64. At the end of the reversal period the only group where a difference between treated males and controls was apparent was the group receiving low melting point wax. However, the·absolute and relative spleen weight was still higher than controls in the female reversal groups that had shown an effect in the main study. Absolute and relative thymus weights were higher than control in all the female groups treated with paraffin wax 64 or micro/paraffin wax mixture. However, the effect was small, and a clear dose response was not
demonstrated for either of the treatments. At the end of the reversal period no difference between treated animals and controls was apparent.
GROSS PATHOLOGY and HISTOPATHOLOGY
The histopathological findings at the end of treatment confirmed the liver, heart and mesenteric lymph node as the primary sites of effect for all of the waxes tested. The presence of lesions in the cervical lymph node in addition to the mesenteric nodes confirms that the effects are not confined to the absorption path of the test articles. The presence of lesions in the mitral valve of the heart confirms the reproducibility of this effect of treatment while the association of birefringent particles with the lesions suggests a direct mechanism for this effect rather than indirect. The histopathological picture at the end of the reversal phase is consistent with the resolution of the lesions resulting from treatment.

Effect levels

Dose descriptor:
NOAEL
Effect level:
other: NOAEL not identified
Sex:
male/female
Basis for effect level:
other: a LOAEL could not be identified because treatment-related effects extended to the lowest dose tested for all three materials.
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Calculated compound intake.

Treatment (% in diet)

Calculated intake (mg/kg/day)

Females

Males

Control

0%

0

0

Paraffin wax 64

0.02%

19.5 ± 0.8

15.4 ± 1.0

Paraffin wax 64

0.2%

193.4 ± 11.7

154.8 ± 9.6

Paraffin wax 64

2.0%

1949.2 ± 78.2

1577.3 ± 83.5

Micro/paraffin wax mixture

0.02%

19.6 ± 0.9

15.3 ± 0.7

Micro/paraffin wax mixture

0.2%

197.6 ± 10.2

154.9 ± 8.9

Micro/paraffin wax mixture

2.0%

1986.2 ± 91.9

1599.2 ± 77.6

Low melting point wax

2.0%

2010.3 ± 77.3

1609.2 ± 84.8

Data tables with specific values for haematology, body weights, organ weights, and other measurement were not provided in the study report, and therefore only qualitative comparisons are reported in this record.

Applicant's summary and conclusion

Conclusions:
A NOAEL could not be determined because adverse effects were noted at the lowest dose (0.02% in diet).
Executive summary:

In a 90 -day oral feeding study, three waxes designated as low melting point wax (LMPW), microcrystalline/paraffin wax mixture (MP), and paraffin 64, intermediate melting point wax (IMPW) were administered via the diet. The tests included a main study, a reversal study and a tissue level study. The reversal group was treated for 90 days followed by 85 days with untreated diet. Group size was 20 rats/sex/untreated control or dose for the main study, 10 rats/sex/the untreated control or high dose (2.0%) for the reversal study, and 5 rats/sex at the high dose only for the tissue level study. Fewer dietary concentration levels were used: IMPW and MP were administered at 0.02, 0.2, and 2.0% and LMPW at 2.0% only. All animals were monitored for weight gain, food uptake and clinical condition throughout the study. An ophthalmic examination was conducted prior to treatment and prior to necropsy on the animals in the main study and those in the reversibility study.

 

There were no effects on food intake, growth rate, or clinical conditions of animals fed paraffin waxes (LMPW, IMPW, or MP). There were some increases in organ weights which included increased spleen and liver weights (0.2 and 2.0% groups). Although some reductions were noted during the reversal period, the weights were still higher in the 2.0% group. Mesenteric lymph node weights were also increased in the high dose group animals, and, like the spleen and liver weights, did not return completely to control weights during the reversal period. There were also changes in haematological parameters with a greater response observed in females. Serum enzymes including alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), and gamma glutamyl transferase (GGT) were elevated in females from the 0.2 and 2% groups treated with paraffin wax (LMPW) and a mix of paraffin and microcrystalline waxes (MP) and from the 2% group treated with intermediate melting point paraffin wax (IMPW) and in males from the 2% LMPW, MP, and IMPW groups.  Serum bilirubin was also elevated in females from the 2% dose group. Histopathological changes in most organs were considered to be consistent with the age of the animals and not treatment-related, but changes in the liver, MLNs, and the cardiac mitral valve were considered to have been treatment-related. The effects were dose-related, more severe in females than males, and greater with LMPW than IMPW and MP.

 

A NOAEL could not be determined because adverse effects were noted at the lowest dose (0.02% in diet).

 

This study received a Klimisch score of 1 and is classified as reliable without restriction. The study was conducted according to Good Laboratory Practices and adhered to OECD guideline 408.