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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

1
Reference substance name:
Hydrogenated rosin alcohols
EC Number:
701-057-0
Cas Number:
2156595-41-2
IUPAC Name:
Hydrogenated rosin alcohols
Test material form:
liquid: viscous
Details on test material:
Sponsor's identification: Technical Hydroabietyl Alcohol
Description: Light amber coloured extremely viscous liquid
Lot number: HR-078
Purity: 100% (UVCB)
Expiry date: 21 December 2012
Storage conditions: Room temperature in the dark
Specific details on test material used for the study:
Test item: Hydrogenated Rosin Alcohols.
Intended use: Industrial use.
Appearance: Colorless tacky resin.
Storage conditions: At ambient temperature (15 to 25°C), in the dark.
Supplier: Sponsor.
Batch number: VR-096
Expiry date: 08 January 2019 - One year from receipt (08 January 2018).
Purity: 100%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 g representative sample was taken from each batch of test item. This sample was placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The CD [Crl:CD (SD)] strain was used since it is a commonly used strain, accepted by regulatory bodies and because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Care and Husbandry
Environmental Control

Rodent facility Limited barrier - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%. Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.
Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage Pre-pairing up to four animals of one sex, Pairing one male and one female , Males after mating up to four animals
Gestation one female
Lactation one female + litter
3.4.3 Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.
Nesting material Paper shavings were provided to each cage from Day 20 after mating and changed at the same frequency as the bedding.
3.4.4 Diet Supply
Diet SDS VRF1 Certified powdered diet.
A sample (250g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30ºC) until finalization of the report. Samples were discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted (See Section 4).
3.4.5 Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The oral route is the most common and is the most appropriate for this testing.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Route Oral, via the diet.
Treated at Constant dietary concentrations (ppm) for each group.
Control (Group 1) Untreated diet of the same batch.
Frequency Continuously.
Diet A record of the usage of the diets was maintained on all occasions when food consumption was measured. This was performed using the initial weight of the diet container and an on-line data check on completion of the feeding procedure to ensure that all cages were fed the correct amount of diet. No significant discrepancy was found.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 100 and 20000 mg/mL were analyzed to assess the stability and homogeneity of the test item in the diet matrix.

Achieved concentration: Samples of each formulation prepared for administration in Week 1 and in the last week of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
n/a for oral studies
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
4 000 ppm
Dose / conc.:
7 500 ppm
Dose / conc.:
15 000 ppm
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Sacrifice and pathology:
See below
Statistics:
See "other information" as the character space limits here won't allow entry in this field.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were considered to be no signs seen that were related to treatment during detailed physical examination and arena observations for animals in the treatment, gestation or lactation period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall body weight gains in males during treatment and females before pairing was low in males at 7500 ppm and females at 15000 ppm (73% and 70%, respectively); and, markedly low for males at 15000 ppm and females at 7500 ppm (61% and 52%, respectively).
During the first week of treatment, group mean body weight gain was markedly low at 15000 ppm (4 g and 1 g compared with 27g and 9g of the Control, for males and females respectively). Thereafter, there was a general improvement in body weight gains, however they remained low for males until Week 4.
Females receiving 15000 ppm commenced gestation with slightly low body weight, when compared to Control (94%), however during the first 2 weeks of gestation (Days 0 to 14) body weight gains were similar to Control. And, although the difference did not attain statistical significance, the overall body weight gain seen in females at 15000 ppm during gestation was slightly low and was primarily due to their performance during Gestation Days 14 to 20 (86% of Control). There was no effect on body weights during gestation for females at 4000 or 7500 ppm.
Body weight gain was unaffected by treatment during lactation. The group mean body weight value for females receiving 15000 ppm when they commenced lactation was slightly low when compared to Control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake was lower than Control for males receiving 7500 or 15000 ppm throughout the treatment period, although statistical significance was only achieved in Weeks 1 (85% of Control at 15000 ppm) or 5 (90% of Control at 7500 ppm).
Prior to pairing, food intake of females receiving 15000 ppm was lower during Week 1 (84 % of Control) or in females at 7500 ppm in Week 2 (82% of Control).
During gestation, food intake of females receiving 7500 or 15000 ppm was lower during gestation days 0 to 14 (86 or 91% of Controls for period), but similar (100 or 96% of Controls for period) during gestation days 14 to 20.
During lactation, food intake of females receiving 15000 ppm was (90%) lower, although the differences did not attain statistical significance.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The hematological investigation revealed a decreased hematocrit value (94%) in males receiving 15000 ppm although there were no corresponding changes in the other erythrocyte indices. In females, haematocrit concentration was also decreased in females receiving 7500 or 15000 ppm (89 or 87% of Control, respectively), with a consequential (since MCHC can be derived from hemaglobin and hematocrit values) increase in mean cell hemaglobin concentration (105 or 104% of Control, respectively), although the difference was offset since the hemaglobin concentration was also reduced (94 or 90%, of Control, respectively).
All differences from controls were minor, confined to one sex or lacked dose-response and were therefore attributed to normal biological variation. Such differences included the slightly low lymphocyte count in males at 15000 ppm (69% of Control), since there was no clear relationship to dose, was not present in females and may have been influenced by two particularly high values in the Control group. The lower basophil count in females at 15000 ppm (33% of Control) or the decreased prothrombin time in treated females (117, 114 or 118% at 4500, 7500 or 15000 ppm) since there was no relationship to dose and no clear effect seen in the males.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was a statistically significantly decrease of phosphorus in males at 15000 ppm and females at 4000, 7500 or 15000 ppm (77% and 56, 72 or 65%, respectively). There was an increase in alanine-amino transferase in males and females at 15000 ppm (141% and 115%, respectively), although statistical significance was only attained in males. There was also a decrease in triglyceride concentrations of males receiving 4000, 7500 or 15000 ppm (65, 58 or 64%, respectively), although this was not present in the females. Lastly, plasma cholesterol concentrations in females receiving 7500 or 15000 ppm were increased (130 or 129%, respectively).
All other differences from controls including those that attained statistical significance, were minor, confined to one sex or lacked dose-response and were therefore attributed to normal biological variation. Such differences included the decrease in bile acids in males (25% of Control), since no corresponding pattern was seen in females. And, the increased albumin (95% of Control) since the change was minor.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity and grip strength were considered unaffected by treatment. There were no consistent intergroup differences in grip strength between males and females.
There was a slightly high value for group mean forelimb grip strength in males receiving 15000 ppm, with the differences attaining statistical significance, however the difference was minor and not present in the females.

Motor activity scores for animals given Hydrogenated Rosin Alcohols were considered to be unaffected by treatment.

Immunological findings:
not examined
Description (incidence and severity):
The analysis of organ weight performed at scheduled termination revealed, when compared with controls, statistically significantly high adjusted liver weights in females at 4000, 7500 or 15000 ppm (123, 138 and 155%, respectively) and at 7500 or 15000 ppm in males (113 or 131%, respectively); the lower adrenal weights in females at 4000, 7500 or 15000 ppm (73, 79 or 76%, respectively); and, the marginally lower adjusted heart weights in females at 7500 (90%) or 15000 ppm (89%).
Statistical significance was attained for thymus weights in males (160% of Control) and females (56% of Control), however there was no clear relationship with dose and the pattern of change differed between the sexes.
All other differences from controls were minor, confined to one sex or lacked dose-response and were therefore attributed to normal biological variation.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic examination performed on Day 13 of lactation revealed distended stomachs of one female at 4000 ppm and one female at 15000 ppm. Due to the pattern and incidence of the finding this distention was not considered related to treatment.
There were no macroscopic changes in males after 5 weeks of treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The microscopic examination performed after 5 weeks of treatment revealed no test item related lesions. The incidence and distribution of all findings were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Effect levels

Dose descriptor:
NOAEL
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Statistics

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including estrous cycles before treatment, pre‑coital interval and gestation indexthe similarity of the data was such that analyses were not considered to be necessary.

All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

The following data types were analyzed at each timepoint separately:

Body weight, using absolute weights and gains over appropriate study periods

Food consumption, over appropriate study periods

Estrous cycles during treatment

Mating performance and fertility

Gestation length

Stage of estrous cycle at termination

Hematology

Blood chemistry

Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index, live birth index and viability index), for before blood sampling study periods

Ano-genital distance, adjusted for pup body weight

Organ weights, both absolute or adjusted for terminal body weight

The following comparisons were performed:

Group 1 vs 2, 3 and 4

 

The following sequence of statistical tests was used for body weight, food consumption, hematology, blood chemistry, implantations, litter size, sex ratio - percentage male, post implantation survival index, ano‑genital distance and organ weight data:

A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pretreatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons usingt-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons the F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1= NMS/EMS which can be compared with standard tables of theF-distribution with 1 and error degrees of freedom. If the F1approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.

 

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pretreatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made. For all other comparisons the H1approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test (Shirley 1977). The test statistic compares the non-monotonicity sums of squares, NRSS, for the deviations of the observed mean ranks from the maximum likelihood mean ranks with the non-parametric equivalent of the error sums of squares, ERSS = N(N+1)/12. The test statistic is H1= NRSS/ERSS which can be compared to standard tables of the χ2-distribution with 1 degree of freedom. If the H1approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.

 

For live birth and viability indices dichotomized to 1 when 100% and 0 otherwise, if the Cochran‑Armitage test (Armitage 1955) was significant at the 5% level, then the direction of the trend was established and one-tailed step-down testing in this direction was performed. If the Cochran-Armitage test was not significant at the 5% level, then a Chi-square test (Armitage et al 2002) was applied. If the Chi-square test was significant at the 5% level, the treatment groups were compared using pairwise comparisons of each dose group against the Control using Fisher’s exact tests (Fisher 1973); otherwise, no further comparisons were made.

For gestation length an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups. If the test was statistically significant (p<0.05), the highest dose group was excluded and the test re-applied. This ‘step-down’ process was repeated until the test was no longer statistically significant (p≥0.05). If the exact version of the Linear-by-linear test could not be calculated (due to the size of the table containing the data), then the asymptotic version was used instead.

For estrous cycles an exact one-tailed (upper-tail) Linear-by-linear test (Cytel 1995) was applied to all groups, using scores appropriate to the severity of the observation assuming 4 day cycles to be normal. The categories were scored as follows: a 4 day cycle was scored as 4, a 4/5 day cycle was scored as 4.5, a 5 day cycle was scored as 5 and irregular and acyclic cycles were scored as 6. If the test was statistically significant (p<0.05), the highest dose group was excluded and the test re-applied. This ‘step-down’ process was repeated until the test was no longer statistically significant (p≥0.05). If the exact version of the Linear‑by‑linear test could not be calculated (due to the size of the table containing the data), then the asymptotic version was used instead.

For number conceiving and number fertile, an exact one-tailed (lower-tail) Cochran-Armitage test (Cytel 1995) was applied to all groups. If the test was statistically significant (p<0.05), the highest dose group was excluded and the test re-applied. This ‘step down’ process was repeated until the test was no longer statistically significant (p≥0.05).

For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights. Similarly, for the litter average ano-genital distance, analysis of covariance was performed using the average pup body weight for each litter as the covariate.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level. The key to the annotation used on the tables that contain statistical results is given below:

l

Data were log transformed for the statistical analysis

 

 

Av

Pretreatment comparison of all groups using Analysis of variance followed by pairwiset-tests

CA

Trend test using Cochran-Armitage test

Ch

Comparison of all groups using Chi-square test

Du

Treated groups compared with Control using Dunnett’s test

Fe

Treated groups compared with Control using Fisher’s Exact test

Lt

Treated groups compared with Control using Linear by Linear tests

Sh

Treated groups compared with Control using Shirley’s test

Wi

Treated groups compared with Control using Williams’ test

 

 

*

p<0.05

**

p<0.01

 

 

Codes placed above the adjusted means indicate that the comparisons were based on adjusted means.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the No-observed-adverse-effect-level (NOAEL) of Hydrogenated Rosin Alcohols for systemic toxicity is 15000 ppm (equivalent to 902 mg/kg/day for males and 1077 mg/kg/day for females) and for reproductive/developmental effects is 7500 ppm (equivalent to 417 mg/kg/day for males and 498 or 984 mg/kg/day for females during gestation or lactation, respectively) based on the reduced offspring body weight.
Executive summary:

The purpose of this study was the assessment of general systemic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine relevant endpoints, with administration of Hydrogenated Rosin Alcohols, for industrial use, by dietary administration for at least five weeks.

Three groups of ten male and ten female rats received Hydrogenated Rosin Alcohols orally, via the diet, at concentrations of 4000, 7500 or 15000 ppm. Males were treated daily for two weeks before pairing, up to necropsy after 35 consecutive days. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, untreated diet of the same batch.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Mean achieved doses for males at 4000, 7500 or 15000 ppm were 240, 417 or 902 mg/kg/day, respectively. For females prior to pairing mean achieved doses were 283, 494 or 1077 mg/kg/day, respectively.

Mean achieved doses for females during gestation were 284, 498 and 1019 mg/kg/day, respectively, and for females during lactation were 610, 984 or 1843 mg/kg/day, respectively.

Analyses of samples for thyroxine (T4) obtained from Main study male animals and F1 offspring on Day 13 of age did not reveal any differences that could be attributed to treatment.

Administration of Hydrogenated Rosin Alcohols at dietary concentrations of 4000, 7500 or 15000 ppm had no adverse effect on clinical condition, sensory reactivity and grip strength, motor activity,hematology,estrous cycles, pre-coital interval, mating performance, fertility and gestation length, macropathology of the adults or micropathology.

For males during treatment, body weight gains and food intake were 73% or 61% of Controls, and for females prior to pairing body weights were 52% or 70% of Controls, at 7500 or 15000 ppm, respectively. Food intake remained low for females during gestation, at 7500 or 15000 ppm (86 or 91% of Controls during gestation days 0 to 14, respectively), and lactation, at 15000 ppm (90%), although reduced body weight gains, at 15000 ppm, was only seen in the later part of gestation (86% of Control from days 14 to 20).

Biochemical examination at the end of the treatment period revealed low phosphorus in males at 15000 ppm and in all groups of treated females (77% and 56, 72 or 65% at 4000, 7500 or 15000 ppm, respectively); an increase in alanine-amino transferase in males and females at 15000 ppm (141% and 115%, respectively); low triglyceride concentrations in treated males (65, 58 or 64% at 4000, 7500 or 15000 ppm, respectively); and, slightly high cholesterol concentrations in females receiving 7500 or 15000 ppm (130 or 129%, respectively). 

At scheduled termination, analysis of organ weights revealed high adjusted liver weights in all treated female groups(between 123 to 155%)and in males at 7500 or 15000 ppm(113 or 131%, respectively), with relationship to treatment.

Implantation counts were slightly low at 7500 or 15000 ppm (88 or 86%) but live litter size was unaffected. There was no effect of parental treatment on offspring clinical signs, sex ratio, offspring body weight on Day 1, survival, nipple counts, ano-genital distances or macropathology. 

Offspring body weight by Day 13 from parents treated at 15000 ppm was lower than Controls due to reduced growth at 19 to 20% lower than Controls.

Based on the results of this study it is concluded that the No-observed-adverse-effect-level (NOAEL) of Hydrogenated Rosin Alcohols for systemic toxicity is 15000 ppm (equivalent to 902 mg/kg/day for males and 1077 mg/kg/day for females) and for reproductive/developmental effects is 7500 ppm (equivalent to 417 mg/kg/day for males and 498 or 984 mg/kg/day for females during gestation or lactation, respectively) based on the reduced offspring body weight.