Reaction mass of 1-methyl-4-(1-methylethylidene)cyclohexyl acetate and p-menth-1-en-8-yl acetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fertility: The NOAEL for fertility for Terpinyl Acetate multi is >=250 mg/kg bw based on read across from Terpineol multi which was tested in an OECD TG 422 effects. In this test effects were seen on male reproductive organs. It was, however, shown that this occurs at gavage dosing but not at dietary dosing and that from 600 mg/kg bw onward the kinetics indicated overloading the metabolic pathway by an increase in AUC. These effects were not seen in a 90 -day inhalation toxicity study where the maximum dose was 400 mg/kg bw when converted from 2230 mg/m3. Also in an older 20 -week dietary study with Terpinyl Acetate alpha no effects were seen on testes at a dose of ca 400 mg/kg bw when converted from diet (10000 ppm). The overall NOAEL for fertility is set to >=250 mg/kg bw because this is the maximum tolerable dose when taking into account the kinetic information.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

Fertility information can be derived from Terpineol multi and for alpha-Terpinyl Acetate subchronic repeated dose information is available. The effects are considered due to gavage dosing as no such effects were seen solely during dietary dosing

Additional information

Below the executive summaries of the source studies are presented and in the developmental section the read across justification is presented. The fertility and developmental effects of the Terpineol multi (OECD 422) which is the main metabolite of Terpinyl Acetate multi (and their respective constituents) is presented in addition to information on fertility from a 20-week repeated dose toxicity study with Alpha-Terpinyl Acetate, major constituent of Terpinyl Acetate multi. The OECD 422 study is presented in detail because of the effects seen in the first study but which have been disregarded based on additional testing in the second study and information on alpha-Terpinyl Acetate.

Alpha-Terpinyl Acetate

In a 20-weeks oral exposure study Osborne-Mendel rats (10/dose/sex) were administered the substance via diet intake at concentrations of 0 (control), 10000, 2500 and 1000 ppm. Animals were then observed for mortality, weight, food intake and general condition. Haematological examinations were made at termination. At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Detailed microscopic examinations was done on the animals from the high dose group and the control group.

No effect on growth or haematology, and no macroscopic or microscopic change in the tissues in the 10000 ppm exposure group were observed. No effect on growth or haematology, and no macroscopic change in the tissues in the 2500 and 1000 ppm exposure groups. No microscopic examination was performed on rats exposed to 2500 and 1000 ppm. According to the Guidance on information and chemical safety assessment R8 table R 8 -17, rats eat 40 to 50 mg/kg bw/day. 10000 ppm is equivalent to 1% in the diet. Consequently, rats exposed to 10000 ppm in the diet consumed between 400 and 500 mg substance per kg body weight per day. Therefore, the NOAEL was calculated to be 400 mg/kg bw.

Under the conditions of the test (OECD TG 408), the NOAEL was determined to be >= 400 mg/kg bw/day for males and females, which is equivalent to 10000 ppm and is the highest dose tested.

Terpineol multi

OECD 422 study (Thacker, 2010a) (this information is also presented in the repeated dose section):

Introduction and methodology: In a GLP study conducted according to OECD TG 422 and in compliance with GLP, three groups, each comprising ten male and ten female rats for the Main (reproductive) phase (exception: five males at control and top dose; additional Recovery phase males were also used for pairing with Main reproductive phase females) and five female rats for the Toxicity phase received Terpineol-Multi at doses of 60, 250 or 750 mg/kg bw/day at a dose volume of 5 mL/kg bw/day. Main phase males and Toxicity phase females were dosed daily for a minimum of five consecutive weeks. An additional ten males and ten females were dosed with the vehicle or at 750 mg/kg/day for five weeks and then given two weeks of recovery before termination. Main phase females were dosed daily for two weeks before pairing, throughout mating, gestation and until Day 6 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology, blood chemistry, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

Results: In the 60 mg/kg/day dose group, one male was found dead on Day 31 of study and one female was killed because of parturition difficulties. In the absence of any other death in the intermediate and high dose groups these deaths are not attributed to the test material. No significant findings were recorded for clinical signs, detailed physical examination and arena observations. Underactive behaviour and unsteady reactions, in males and females were observed briefly during Week 1 in animals receiving 750 mg/kg/day and dose‑related increases in post‑dosing salivation and chin rubbing were seen. Behavioural testing during Week 5 of dosing, including sensory reactivity findings, grip strength values and motor activity scores showed no differences considered to be associated with exposure to the test material. There were no clear effects on bodyweight in males or unmated females receiving up to 750 mg/kg/day. Males receiving 750 mg/kg/day showed lower overall weight gain (Week 0-5) compared with Control. Bodyweight during the recovery phase was similar to Controls. Bodyweight and bodyweight gain were unaffected during gestation. During lactation females receiving 250 mg/kg/day showed lower weight gain than Controls. There were no adverse effects on food consumption in males, unmated females or females during gestation and lactation but visual assessment of water consumption indicated that males and females receiving 750 mg/kg/day were consuming more water than the Controls during the dosing period. Among the toxicity subgroup animals there were no clinically significant effects of Terpineol-Multi upon haematology parameters. Females showed slight anaemia but males were essentially unaffected. At the end of the two week recovery period, no intergroup differences were present in females whereas haematocrit, haemoglobin and red blood cell count were slightly decreased in males. At 750 mg.kg/day, urea and creatinine levels were significantly higher than Controls in females and slightly high in males. Glucose plasma levels were significantly higher than Control in females dosed at 250 and 750 mg/kg/day and marginally high in males. Potassium levels were significantly higher than Control in males and females receiving 750 mg/kg/day. All the above discussed parameters, except for urea and creatinine, showed complete recovery after 2 week recovery period. There were no effects of Terpineol-Multi on oestrous cycles, precoital interval or mating. Gestation length was within the normal range but there was a small increase in the numbers of animals at 250 mg/kg/day having longer (23 day) gestation periods. At dose levels up to and including 250 mg/kg/day there were no effects of the test material on the number of implantations, post implantation survival index, live birth index, viability index and lactation index. Male and female offspring bodyweights were not adversely affected by Terpineol-Multi. At 750 mg/kg/day, relative liver weights were significantly higher than Control in males and females and relative kidney weights were significantly higher than Control in males. Testis weight was markedly low in males receiving 750 mg/kg/day and there was also an indication of low epididymal weights at this dose. Liver and kidney weights returned to normal after two weeks when the animals did not receive Terpineol-Multi but testis and epididymal weights showed no evidence of recovery. Adaptive centrilobular hepatocyte hypertrophy in the liver of females dosed with Terpineol-Multi at 750 mg/kg/day was not present after 2 weeks recovery and histopathological findings in the kidneys of males receiving 250 and 750 mg/kg/day also resolved after the end of dosing. At 750 mg/kg/day, reduced numbers or complete absence of spermatozoa, accompanied by the presence of degenerate spermatogenic cells in duct(s) were observed in the epididymides and were still present following the 2‑week recovery period. Spermatocele granuloma(ta) that were seen in two males receiving 750 mg/kg/day and one receiving 60 mg/kg/day were not seen at the end of the recovery period. The significance of this change in the single male receiving 60 mg/kg/day is uncertain as spermatocele granuloma(ta) can occur spontaneously in rats of this age and considering the absence of other degenerative changes in the testes or epididymides of this animal. Moderate to severe seminiferous tubular atrophy/degeneration was seen in the testes of all animals dosed with Terpineol-Multi at 750 mg/kg/day, accompanied by minimal to moderate spermatid giant cells and minimal to slight seminiferous tubular vacuolation. Similar findings were still evident following the 2‑week recovery period but at a lower incidence and severity suggesting a degree of recovery.

Based on the findings in this study, the systemic NOAEL for males and unmated females was 250 mg/kg/day, the NOAEL for maternal and developmental toxicity was at least 250 mg/kg/day.

Investigatory study (Thacker, 2010c):

In this study, the toxicity of Terpineol-Multi to the male reproductive system was examined when administered by dietary or oral gavage routes. Three groups of Crl:CD(SD) male rats (five/dose) were dosed daily with Terpineol-Multi by dietary and/or oral gavage routes at the following doses: group 1: dietary 7500 ppm + supplementary gavage dose 300 mg/kg/day, group 2: dietary 10000 ppm + supplementary gavage dose 150 mg/kg/day, group 3: Terpineol-Multi at 750 mg/kg/day by gavage only. During the study, data was recorded on mortality, clinical condition, bodyweight and food consumption. Surviving animals were subjected to a detailed sperm analysis. Testes (L&R), epididymis (L&R), prostate and seminal vesicles were weighed at necropsy and tissues of right testes and epididymis were fixed for histopathological examination.

During week 1 of the study, Group 1 and 2 animals consumed less diet than expected; From Day 11 Group 1 animals, receiving 7500 ppm, also received two daily doses of 150 mg/kg/b.i.d. four to five hours apart and average intake was boosted to 663 mg/kg/day whilst Group 2 animals, receiving 10000 ppm, also received a single daily dose of 150 mg/kg boosting intake to 678 mg/kg/day. Clinical signs were generally minimal. Dosing signs for Group 3 animals included salivation and chin rubbing. Food consumption for Group 1 and 2 animals receiving the test material in the diet was low throughout the study; this was attributed to the palatability of the test material. Bodyweight gains of these animals were also low. Food consumption and bodyweight changes of Group 3 animals were considered to be not adversely affected by the test material. Necropsy data indicated that decreases in reproductive organ weights and changes to macroscopic appearance were most marked in the animals receiving Terpineol-Multi at 750 mg/kg/day. Occasional animals (1/5 and 1/5 in each of the other groups 1 and 2 respectively) also showed changes to testicular and/or epididymal tissue appearances and/or weights. Sperm analysis showed that motile sperm with normal morphology were present in 4/5 males of Group 2 and 1/5 males of Group 1. The outliers in each group were at the extreme of achieved overall exposure for the group suggesting that absolute exposure was important, although the route of exposure and consequently potential to exceed threshold levels was of greater significance. Microscopic examination indicated there were relatively fewer changes in the testes and epididymides in the animals which were given Terpineol-Multi by the dietary route with oral gavage supplementation (Groups 1 and 2), whereas there were significant changes in those which received it solely by oral gavage (Group 3).

The results of dietary administration suggest that exposure via the dietary route of administration reduces the testicular and sperm toxicity of the test material compared to dosing by oral gavage. The results of this study, in part, support the hypothesis that a high peak plasma level is necessary to induce the observed toxic effects. The possible MoA is discussed in the repeated dose MoA section.

Conclusion

Terpinyl Acetate multi has no effects on fertility at the highest dose tested resulting in a NOAEL of 250 mg/kg bw because this is the maximum tolerable dose for Terpineol multi. For Terpineol multi testicular atrophy was seen at 750 mg/kg shown to be due to gavage dosing. No such effects were seen for Terpinyl Acetate alpha at 400 mg/kg bw while testes weight was recorded. Therefore the NOAEL >=250 mg/kg bw can be considered for Terpineol Acetate multi.

Reproductive toxicity of Terpinyl Acetate multi using read across from Terpineol multi

Introduction and hypothesis for the analogue approach

The constituents of Terpinyl Acetate multi have a cyclohexyl ring with an unsaturated bond with a methyl group and a tertiary acetate attached on the para-position. For Terpinyl Acetate multi insufficient reproductive dose toxicity information is available.

In accordance with Article 13 of REACH, lacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing the reproductive toxicity of Terpinyl Acetate multi, the analogue approach is selected because for the key metabolite reproductive toxicity information is available which can be used for read across. This type of analogue approach reflects the RAAF-1 approach: (Bio)transformation to a common metabolite.

Hypothesis considering the outcome of the read across: Terpinyl Acetate multi has the same reproductive toxicity as Terpineol multi resulting in the same effects and NOAELbecause Terpinyl acetate will not reach the systemic circulation. The acetate will be metabolized into the alcohol and/or its ring will become similarly oxidised/hydroxylated as Terpineol multi. Conversion considering molecular weight is not needed because Terpinyl Acetate multi has a higher molecular weight and the results for Terpineol multi can be considered conservative.

The hypothesis considering the adverse outcome pathway and key eventsare that 1) Terpinyl Acetate multi will be metabolised into Terpineol multi and other hydroxylated products; 2) Metabolic overload can occur at > 600 mg/kg bw Terpineol and; 3) The toxicity can be seen by liver effects and testicular atrophy.

Available kinetic experimental information: The Terpinyl Acetate multi plasma DT50 of ca. 50 minutes and its DT50 in acidic media of ca 10 minutes are used to predict the absence of Terpinyl Acetate in the systemic circulation (key event 1, See IUCLID Toxicokinetic section). No metabolites or degradation products were seen in the chromatograms including Terpineol, which is likely a limitation in the detection methodology. This study receives Klimisch score 2 because it is not a GLP study and information on degradation products is missing. Therefore, for further reasoning of this absence of the acetate and formation of the alcohol, metabolism data on Linalyl acetate is used for support.

Available reproductive toxicity information, fertility: The reproductive toxicity of Terpineol multi is determined according to OECD TG 422 and receives reliability 1. The substance caused non adverse liver effects and testicular toxicity at 750 mg/kg bw and the NOAEL for repeated and reproductive toxicity was set at >=250 mg/kg bw,because it was shown that this effects was due to gavage dosing; it was not seen in dietary dosing at the same dose. In the 90-day repeated dose inhalation toxicity test no adverse effects including on gonads were seen at 2230 mg/m3 (OECD TG 413). The NOAEL of this inhalation study can be converted to ca 400 mg/kg bw Terpineol (1 mg/kg bw is 0.0052 mg/l air). The bridging study between Terpineol multi and Terpinyl Acetate multi is the 20-wk dietary study from Hagan (1967) with Terpinyl Acetate alpha,the key constituent of Terpinyl Acetate Multi, in which liver and testes were weighed and no effects were seen at the highest dose tested of 400 mg/kg bw (Klimisch 2, non OECD guideline and non-GLP, conversion from 10000 ppm to 400 mg/kg bw according to ECHA guidance, R8, Table R8-17).

Available reproductive toxicity information, developmental toxicity: Terpineol multi did not show developmental toxicity effects in rat in an OECD TG 414 study resulting in a NOAEL of 600 mg/kg bw.

2. Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemicals are shown in the data matrix.

3. Purity / Impurities

In table 1 the constituents of Terpinyl Acetate multi are presented as well as Terpineol multi to show the overlap in concentrations between constituents in the substances.

Table 1: The constituents of Terpineol multi and Terpinyl Acetate multi

Terpinoids in %	Alpha-Terpineol	Gamma-Terpineol	cis-beta-Terpineol	trans-beta-Terpineol	Alpha-Terpinyl Acetate	Gamma-Terpinyl Acetate	cis-beta- Terpinyl Acetate	trans-beta Terpinyl Acetate
Cas nu	98-55-5	586-81-2	138-87-4Generic	138-87-4, Generic	80-26-2	10235-63-9	2777-47-3	59632-85-8
Terpineol multi	60	29	9	3	0	0	0	0
Terpinyl Acetate multi	0	0	0	0	64	20	7	4

The target substance Terpinyl Acetate multi consists of Terpinyl Acetate alpha and Terpinyl Acetate gamma, with Terpinyl Acetate beta (trans and cis) as impurities. In Terpineol multi a very similar distribution is present for the alcohols. This means that once the Terpinyl Acetate multi conversion to the alcohols are sufficiently characterised it can be assumed that all constituents are sufficiently addressed.

4. Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group or when there is a common metabolite. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation, which is presented below.

Analogue justification: For Terpinyl Acetate multi, Terpineol multi is selected as analogue because the constituents of Terpineol multi are the key metabolites of the constituents of Terpinyl Acetate multi. For Terpineol multi the required information is available. The biotransformation of the alkene terpinoid Linalyl acetate is used to support the read-across justification from the acetate to the alcohol. The key constituent of Terpinyl Acetate multi is Terpinyl Acetate alpha for which a 20-week repeated dose study will be used as bridging study to support the read across, because this 20-week study is insufficientdocumented (and some current parameters may be missed)to be used as a stand-alone study.

Structural similarities and differences: The constituents of Terpinyl Acetate multi and Terpineol multi have the same non-saturated cyclohexyl ring, with a methyl group attached to the unsaturated bond. At the opposite side (C4 position) two methyl group and a tertiary acetate or a tertiary alcohol is attached, respectively.

Toxico-kinetic, Absorption: The similarities considering absorption between the acetate and the alcohol are less relevant because the read across approach is applicable after the absorption step. For completeness both the acetate and alcohol are fully absorbed based on their physico-chemical characteristics with MW < 200 and log Kow values between 2-7 (see Toxico-kinetic section, and data matrix).

Metabolism: Terpineol Acetate multi will be hydrolysed and/or cleaved by carboxyl esterases in the gastro-intestinal tract, the liver and blood as presented by e.g. Belsito et al. (2008) for cyclic acetates and by Wu et al. (2010) for Terpinyl acetate. To verify this degradation of the acetate experimental tests were performed in which 50% of the Terpinyl acetate multi was cleaved in ca. 10 minutes in aqueous solutions with pH 2, 4 and 7. In plasma the half-life was somewhat longer ca. 50 minutes. In the plasma experiment both Terpinyl Acetate alpha and gamma decreased in a similar manner (see toxico-kinetic section for details).

Though the decrease of the Terpinyl Acetate was seen, no formation of any metabolite was seen including the alcohol. This is considered a limitation of the GC-MS MS method used or how the method was applied. To support the acetate-alcohol conversion there is experimental metabolism information on Linalyl Acetate in which Linalool is detected. Terpinyl Acetate is very similar to Linalyl acetate. The only difference is that Terpinyl Acetate has a closed ring and Linalyl acetate has a straight alkene chain to with a tertiary ester is attached (Table 2). The formation of Linalool as identified by Hall (1979) supports the formation of Terpineol from Terpinyl Acetate (EFSA (2011) and kinetic section). Therefore, the read across of Terpinyl Acetate multi from Terpineol can be pursued. Further metabolisation work is ongoing to detect the metabolic products of Terpinyl Acetate multirepeated dose and reproductive toxicity (see REACH dissemination tool).

After the cleavage of the ester and/or hydroxylation of the ring, in Phase 2 of the metabolisation, the alcohol will be glucuronidated for excretion (WHO, 1999, WHO 2000, RIFM, 2016).

Table 2Half-life (DT50 or T ½) are shown for Terpinyl Acetate multi and Linalyl Acetate based on experimental information

	Terpinyl Acetate multi; T1/2 minutes (k elimination in brackets)	Linalyl acetate; T1/2 minutes (k elimination in brackets)
Cas no.	8000-41-7 (generic)	115-95-7
Structural features of metabolism
MW	196	196
Acidic artificial gastric juice	8.8 (water, pH2)	5.5
Pancreatic fluids	7.5 (water, pH4)	52.5
Neutral gastric fluids	10.3 (water, pH7)	121
Intestinal mucosa, blood and liver	51 (plasma, k=0.0137)	T1/2 not reported (k=0.01 to 0.0055)
Reference	IFF, 2020 (CRL)	Hall, 1979 Unpublished report; As sited in EFSA and WHO, 2006.

Toxico-dynamics: Terpinyl Acetate multi may have the same liver effects and testicular atrophy effects as in case overloading of the metabolic pathway occurs.

For Terpineol it was shown that at gavage doses up to 600 mg/kg bw the metabolic pathway can keep up linearly with increases in gavage doses but at higher peak doses (750 mg/kg bw)the AUC increased disproportionally, which is a sign of overloading of this metabolic pathway. As a result, toxic metabolite(s) become available in the systemic circulation (ECHA Terpineol multi dossier). That 600 mg/kg bw is indeed a cut off is supported with the Terpineol multi 90-day inhalation study. Absence of testicular toxicity were found at ca 2230 mg/m3, which can be converted to 400 mg/kg bw using a respiration rate for rat of 0.38 and taking into account 6/24 hours and 5/7 days weekly exposure and 100% absorption.

The 20-week dietary Terpinyl Acetate alpha of Hagan (1967) can be a bridging study between Terpinyl Acetate multi and Terpineol multi. It supports the absence of overloading the metabolic pathway up to 400 mg/kg bw (the highest dietary dose in this Hagan study, 10000 ppm) and absence of testicular effects.

Similarities in results for toxicological endpoints other than reproductive toxicity: There are no acute systemic toxicity differences and no genotoxic differences between Terpinyl Acetate multi and Terpineol multi: these substances are non-genotoxic, while they show cytotoxicity at fairly low concentrations.

Uncertainty of the prediction: There is uncertainty on the exact quantitative metabolic pathway of Terpinyl Acetate multi to Terpineol multi. The absence of Terpinyl Acetate multi in the systemic circulation has been experimentally shown in plasma and acidic conditions. However, no degradation products could be identified due to methodological limitations of the test set-up. Therefore, information on Linalyl acetate is used, which is also a tertiary terpinyl acetate and metabolises into Linalool. Further study is currently initiated to find the metabolites of Terpinyl Acetate multi.

The available information on in vitro and in vivo metabolism studies of Terpineol (alpha and multi) indicated that at high gavage dosing an overload mechanism can occur (RIFM (2016)and Terpineol multi dossier on ECHA dissemination site, respectively). When testing Terpinyl Acetate multi at similar high gavage doses, this may occur as well. The dose for Terpinyl Acetate multi would be 955 mg/kg bw when using a conversion from Terpineol (750 mg/kg bw effect dose Terpineol with a MW of 154, result in 955 mg/kg bw Terpinyl Acetate with a MW of 196). No testicular effects were seen for Terpinyl Acetate alpha in the 20-week dietary study at ca 400 mg/kg bw, which is indeed below the critical value of 955 mg/kg bw Terpinyl Acetatewhen using LOAEL conversion from Terpineol. Though there is uncertainty considering the exact NOAEL for testicular effects, however it is likely that the NOAEL of >=250 mg/kg bw from Terpineol can be considered conservative for Terpinyl Acetate multi.

5. Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the Data Matrix.

6. Conclusions for reproductive toxicity

For Terpinyl Acetate multi insufficient reproductive toxicity information is available. For the key metabolites (constituents of Terpineol multi) such data is present, which can be used for read across. This analogue approach is adequate and reliably presented in the present document and the result can be used for C&L and/or risk assessment.

For assessing the reproductive toxicity of Terpinyl Acetate multi, the absence of Terpinyl Acetate in the systemic circulation and the formation of Terpineol multi are the basis for this assessment. Terpinyl Acetate multi has half-lives in plasma of ca 50 minutes and in aqueous environments of ca 10 minutes. This means that after passage of stomach, gastro-intestinal tract and liver no Terpinyl Acetate multi will be present in the systemic circulation. In absence of any degradation product, additional metabolism information from Linalyl acetate (the open version (alkene tertiary acetate) of Terpinyl acetate) is used in which the formation of Linalool is shown. Therefore, the constituents of Terpinyl Acetate multi are expected to turn into the constituents of Terpineol multi.

For Terpineol multi there is reproductive toxicity data: Repeated dose Reproscreen study according to OECD TG 422 and a 90-day inhalation study according to OECD TG 413. The read across between these two is bridged with repeated dose toxicity data from Terpinyl Acetate alpha (20-week dietary study including histopathology of key organs a.o. testes). The available information on Terpineol multi indicates liver effect and testicular atrophy at high gavage doses (750 mg/kg bw). Available kinetic information on Terpineol multi indicates overload of the metabolic pathway at doses >=600 mg/kg bw. Using all this information the NOAEL for Terpineol and Terpinyl Acetate multi constituents would results in a NOAEL >= 600 mg/kg bw because toxicity caused by saturation of the enzymes in the metabolic pathway do not need to be considered for toxicity. In view of a NOAEL for testicular toxicity derived in the OECD TG 422 of >=250 mg/kg bw, this result will be used for the risk assessment being the lowest and therefore conservative value.

In a rat developmental toxicity study with Terpineol multi no effects were seen. These will be directly used for read across to Terpinyl acetate multi resulting in a NOAEL of 600 mg/kg bw.

Final conclusion on hazard:Terpineol Acetate multi has a NOAEL of >=250 mg/kg bw for repeated dose toxicity and fertility and of 600 mg/kg bw for developmental toxicity.

Data matrix for the read across to Terpinyl acetate multi from Terpineol multi with support from Terpinyl Acetate alpha.

Common names	Terpinyl Acetate multi	Terpineol multi	Terpinyl Acetate alpha
	Target	Source	Source supporting
Chemical structures		See Terpineol alpha as key const.	See Terpinyl Acetate multi
CAS no	Not relevant	(8007-35-0)	80-26-2
ECHA registration	904-693-9	701-188-3	201-265-7
Molecular weight	196	154	196
Physico-chemical data		ECHA site	ECHA site
Appearance	Liquid	Liquid	Liquid
Water solubility, mg/l	36	2540	36
Log Kow	4.4	2.6	4.4
Human health endpoints
Toxico-kinetic, metabolism	Read across from Linalyl Acetate	Key metabolite of Terpinyl Acetate multi constituents
Acute oral tox in mg/kg bw	Read across from Terpinyl Acetate alpha	2000 (OECD TG 401)	5075 (Similar to OECD 401)
Acute dermal tox in mg/kg bw
Genotoxicity – Ames test (OECD TG 471)	Negative	Negative
Genotoxicity-cytogenicity	Negative	Negative
Genotoxicity_MLA	Negative	Negative
Repeated dose toxicity
28-day Repeated dose toxicity gavage mg/kg bw	Read across from Terpineol multi	NOAEL >=250 (OECD TG 422)
90-day Inhalation study	Read across from Terpineol multi	2230 mg/m3 Ca 400 mg/kg bw (OECD TG 413)
20-week dietary study			NOAEL >=400 mg/kg bw (eqv OECD TG 408)
Fertility
28-day Repeated dose/ Reprosceen study via gavage mg/kg bw	Read across from Terpineol multi	NOAEL >=250 (OECD TG 422)
20-wk dietary study mg/kg bw			No effects on gonads at 400 mg/kg b(eqv OECDTG408)
Developmental toxicity
Developmental toxicity in mg/kg bw	Read across from Terpineol multi	600 (OECDTG414)

References

Belsito, D., Bickers, D., Bruze, M., Calow, P., Greim, H., Hanifin, J.M., Rogers, A.E., Saurat, J.H., Sipes, I.G., Tagami, H., 2008, A toxicologic and dermatologic assessment of cyclic acetates when used as fragrance ingredients, Food and Chemical Toxicology 46, Suppl 12:S1-27.

EFSA, 2011,https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2011.1847

RIFM, 2016, Alpha Terpineol: Metabolism in rats after single oral (gavage) exposure, unpublished report, RIFM no 70825.

WHO, 1999, Food additive series 42, 1999, Evaluation of certain food additives, Aliphatic acyclic and alicyclic terpenoid tertiary alcohols and structurally related compounds,http://www.inchem.org/documents/jecfa/jecmono/v042je17.htm

WHO, 2000, Evaluation of certain food additives, Technical Report Series 891, page 51-54,http://whqlibdoc.who.int/trs/WHO_TRS_891.pdf.

Wu, S., Blackburn, K., Amburgery, J., Jaworska, J., and Federle, T., 2010, A framework for using structural, reactivity, metabolic and physico-chemical similarity to evaluate the suitability of analogs for SAR-based toxicological assessments, Regul. Toxicol. Pharmacol., 56, 67-81.

 

Effects on developmental toxicity

Description of key information

The developmental toxicity of Terpinyl Acetate multi has been derived based on read across from Terpineol multi for which a rat developmental toxicity study (OECD 414) was performed. In this study no effects were seen up to 600 mg/kg bw, highest dose tested. This is supported by a repeated dose (oral gavage) toxicity study with reproductive/developmental toxicity screening test (OECD 422) of Terpineol multi in which no developmental toxicity effects were seen at 750 mg/kg bw, the highest dose in this study.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Additional information

For Terpinyl Acetate multi no developmental toxicity data are available. Therefore data (OECD 414 and OECD 422) from Terpineol multi will be used. Below the executive summaries of the source studies is presented. The read across is included in the fertility section above.

Terpineol multi: OECD 414

In a GLP-compliant prenatal developmental toxicity study performed according to OECD guideline 414, the test substance diluted in corn oil was administered by gavage to groups of mated female Sprague-Dawley rats (20 mated females/dose) at the dose levels of 0, 60, 200, 600 mg /kg bw/ day from Days 6 to 19 after mating. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Detailed observations were recorded daily at the following times in relation to dose administration. A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health. The weight of each adult was recorded on Days 0, 3 and then daily from Days 6 to 20 after mating. The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating. On Day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The gravid uterine weight, number of implantations, live and dead fetuses, early and late resorptions and corpora lutea were recorded. Gross evaluation of the placenta was also performed. Fetuses were sexed, weighed and examined for external, soft tissue and skeletal malformations. With the exception of one female in the 200 mg/kg bw/day group (No. 52) which had a total litter resorption, all females were found to be pregnant with live young at scheduled termination on Day 20 of gestation. No mortality was observed. At scheduled termination on Day 20 of gestation, the adjusted mean liver weight of females receiving 600 mg/kg bw/day was significantly higher than Control (1.10X Control). There were no treatment-related macroscopic abnormalities detected. No relevant clinical signs or signs of reaction to treatment were noted in treated females. Females receiving 60 or 200 mg/kg bw/day showed no treatment-related changes in clinical condition, body weight performance, food intake, liver weight or macropathology. There was no effect of maternal treatment with the test substance at any dose level investigated on litter data. Sex ratio, as assessed by the percentage of males per litter, was generally comparable in all groups and in line with expectations. Embryo-fetal growth was slightly reduced by maternal treatment at 600 mg/kg bw/day. It was considered that there was no adverse effect of maternal treatment on embryo-fetal development; the incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment with the test substance. In the 600 mg/kg bw/day group there was a slightly higher incidence of incompletely ossified or unossified 5th and/or 6th sternebrae compared to concurrent control and the Historical Control Data range. It was considered that this minor finding did not constitute an adverse effect on development. On the basis of the results obtained in this study, the dosage of 600 mg/kg bw/day was considered to be the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity.

Terpineol multi: OECD 422

There is a combined repeated dose toxicity study with reproduction / developmental toxicity screening available (OECD TG 422) for Terpineol multi, which is summarised in the fertility section.

The read across justification is presented in the fertility section.

Justification for classification or non-classification

Fertility: Terpineol Acetate multi has a NOAEL for fertility of 400 mg/kg bw based on a study of alpha-Terpinyl Acetate being its main constituent which is supported by all information available from Terpineol multi. This is because the effects seen during gavage dosing in the OECD TG 422 are considered secondary effects as mentioned in CLP section 3.7.2.2.1 “Classification is made on the basis of the appropriate criteria, outlined above, and an assessment of the total weight of evidence (see 1.1.1). Classification as a reproductive toxicant is intended to be used for substances which have an intrinsic, specific property to produce an adverse effect on reproduction and substances shall not be so classified if such an effect is produced solely as a non-specific secondary consequence of other toxic effects”. Therefore, for Terpinyl Acetate multi classification and labelling is not needed for this endpoint according to EU CLP (EC No. 1272/2008 and its amendments).

Developmental toxicity: The information from Terpineol multi derived from the OECD TG 414 and OECD TG 422 can be used for screening for developmental toxicity. This information did not result in classification and labelling for developmental toxicity. In the absence of e.g. structural alerts and absence of genotoxicity no other developmental toxicity is expected. Therefore, Terpinyl Acetate multi does not have to be classified and labelled for developmental toxicity according to according to EU CLP Regulation (EC No. 1272/2008 and its amendments).