Registration Dossier

Administrative data

Workers - Hazard via inhalation route

Systemic effects

Long term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
12.7 mg/m³
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
Overall assessment factor (AF):
6
Modified dose descriptor starting point:
NOAEC
DNEL value:
63.5 mg/m³
Acute/short term exposure
DNEL related information

Local effects

Acute/short term exposure
DNEL related information

Workers - Hazard via dermal route

Systemic effects

Long term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
6 mg/kg bw/day
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
Overall assessment factor (AF):
24
Modified dose descriptor starting point:
NOAEL
DNEL value:
144 mg/kg bw/day
Acute/short term exposure
DNEL related information

Workers - Hazard for the eyes

Local effects

Hazard assessment conclusion:
no hazard identified

Additional information - workers

In the 28 day range finding study 5 animals per sex and dose were administered to 1000, 5000 and 15000 ppm (corresponding to ca. 80-100, 360-510 and 1090-1260 mg/kg bw/day). The observed effects were changes in organ weights and food consumption, so that the NOAEL was found to be 80-100 mg/kg bw/day.

Repeated dose toxicity was analyzed in a subchronic 90-days study performed according to OECD Guideline 408 (BASF AG, 2003, 2004). In the subchronic study the compound was administered to groups of 10 male and 10 female Wistar rats at dietary concentrations of 0, 100, 1000 and 10000 ppm (corresponding to 7 and 8 mg/kg bw/day, 72 and 83 mg/kg bw/day or 720 and 801 mg/kg bw/day for males and females, respectively) for 3 months. Substance related effects in liver, kidney and the thyroid gland were detected, including central and peripheral hypertrophy of hepatocytes, decreased thyroxine as well as changes in urinary parameters. Effects observed on reproductive organs were significantly increased absolute and relative weight of the testes and relative weight increase of epididymides in the high dose males, though no histological correlates were detected. The NOEL under the conditions of the present study was 100 ppm for both sexes (about 7 and 8 mg/kg bw/d for males and females). Based on adaptive liver effects in both sexes and minor urine findings in males the NOAEL was 1000 ppm (72 and 83 mg/kg bw/day for males and females, respectively). The LOAEL was found to be 10 000 ppm (720 and 801 mg/kg bw/day for males and females) due to liver, kidney and thyroid findings in both sexes.

In two other studies rats were administered for 5 or 90 days, where a NOAEL of 10 mg/kg bw/day was found after the 90 day application (Hoffmann-LaRoche, 1975; Ford, 1983). However, both studies showed clear deficits in the protocol or given data, so that their results could not be taken into account for evaluation.

 

No adverse effects on reproduction were found in a two-generation study, when 8-10 mg/kg bw/day of the structural analogue mixed ionone isomers (CAS 8013-90-9) were administered to parental rats for 8 months (Sporn, 1963). The F1 generation (offspring) were allowed to reach maturity and were then treated with 15 mg/kg of ionone prior to being subject to reproductive toxicity testing.

                                                                                                                                   

Developmental toxicity was evaluated in a study performed according to OECD Guideline 414 (BASF AG, 2004). Trans-β-ionone was administered as a solution in olive oil to 25 "time-mated" female Wistar rats/group by stomach tube at doses of 25, 100 and 400 mg/kg bw on day 6 through day 19 post coitum (p.c.). As a result, administration of 400 mg/kg bw elicited substance-induced effects on the dams including signs of maternal toxicity like reduced body weight gain (-29%). The dosage of 100 mg/kg bw/day resulted in some substance-related findings (i.e. temporary salivation, marginally increased liver weights), which are not considered to be adverse, but mirror some adaptive responses of the animals. The test substance had no influence on gestational parameters and did not induce adverse signs of developmental toxicity or teratogenic effects at all dose levels. The NOAEL for maternal toxicity has been set at 100 mg/kg bw/day and ≥ 400 mg/kg bw/day for developmental toxicity.

Information for prenatal developmental toxicity of trans-β-ionone is available on a second species, i.e. the rabbit. In a study performed according to OECD Guideline 414 and GLP (BASF SE, 2014) 22 female New Zealand White rabbits/group received trans-β-ionone by inclusion in the diet on day 6 through day 29 p.c. at target dose levels of 17, 50 and 200 mg/kg bw/day. Following dietary treatment at 200 mg/kg bw/day, reduced food consumption (absolute and relative to body weight) were noted during almost the entire treatment period. In addition, reduced body weights, lower body weight gain and/or body weight loss were recorded. The treatment did not result in mortality or gross findings at necropsy. No maternal toxicity was observed in the 17 and 50 mg/kg bw/day groups.

Fetal body weights were slightly lower in males and females at 200 mg/kg bw/day, reaching statistical significance for males only. This change was considered to be secondary to the reduced food intake and markedly decreased body weight gain of the dams. No toxicologically relevant effects on viability, litter size or sex ratio were noted up to 200 mg/kg bw/day. The incidence of unossified metacarpals and/or metatarsals was slightly higher in the fetuses at 200 mg/kg bw/day, reflecting the slightly lower fetal weights observed in this dose group. The maternal and developmental NOAEL for trans-β-ionone was established as being 50 mg/kg bw/day.

As the experimental exposure of a study according to Guideline OECD 414 adequately covered the pregnancy of the species under investigation an AF for exposure duration is not necessary.

 

Therefore, the respective NOAEL of 72 mg/kg bw/day for female rats in the 90-days toxicity study has been taken as conservative point of departure for the respective systemic DNELs derived, which covers findings observed in the subchronic repeated dose and developmental toxicity study.

 

Route to route extrapolation:

On the basis of the low vapour pressure (0.072 hPa), the exposure with trans-β-ionone via inhalation as a vapour is low. According to Chapter R.8 of REACH Guidance on information requirements and chemical safety assessment, it is proposed in the absence of route-specific information to include a default factor in the case of inhalation-to-oral extrapolation, assuming 50% oral and 100% inhalation absorption.

 

No experimental data on dermal absorption of trans-β-ionone are available. However data from thestructurally similar substance methyl-ionone (CAS 1335-46-2) can be used for read-across.Based on comparable physicochemical properties(M = 192 – 206 g/mol, density = 0.93 – 0.95 g/cm3, logPow= 4 – 5, water solubility < 1 g/l, vapour pressure < 10 Pa) both substances are considered to show a limitedbioavailability via the dermal route. In addition both substances are of low acute toxicity(LD50(dermal and oral) > 5000 mg/kg bw).For the above mentioned reasons, dermal adsorption of both substances is considered to be sufficiently similar to support read-across.

In anin vitrodermal penetration/permeability study, only 0.7% or undetectable amounts of methyl ionone (mixture of isomers) were recovered in the fluid beneath the skin preparations of rats and pigs, respectively, 6 h after application of a 3000 µg dose (600 µg/cm² over 5 cm² of skin) (RIFM, 1984a). In this study, approximately 50% (rat) and 10% (pig) of methyl ionone14C penetrated into, but not through the epidermis and dermis, while another 30% was lost to evaporation. (Belsito, 2007)

Skin penetration potential through human skin is generally much closer to that of porcine skin than to that of rat skin. The morphology of porcine skin corresponds much better to that of human skin than that of fur bearing animals, which presents numerous hair follicles. Hair follicles act as shunt ways of resorption and hence, chemical substances penetrate into or through fur bearing skin much easier. Van Ravenzwaay and Leibold (2004) have compared the differences in absorption for a large number of chemicals and found the dermal absorption through rat skin is generally at least 2.3 times greater than through human skin. Taking this information together with the values of the in vitro study (50% penetration into rat skin and 10% penetration into porcine skin, but no penetration through either kind of skin), a maximal penetration of ca. 20% into but not through the skin might be assumed. A worst case of 50% penetration through human skin is assumed fortrans-β-ionone, although realistic values may be considerably lower. Hence, an assessment factor of 0.5 was chosen.

 

Substance specific assessment factor for remaining differences

Although ‘residual’ interspecies variability may remain following allometric scaling, this is largely accounted for in the assessment factors proposed for intraspecies variability, i.e. reflecting the interdependency of inter- and intraspecies assessment factors (Calabrese and Gilbert, 1993).

Furthermore, within the ERASM project, it was suggested that a factor of 2.5 for ‘remaining‘ interspecies differences may be questionable as a standard procedure (Escher and Mangelsdorf, 2009; Batkeet al, 2010; Bitsch et al, 2006). The comparison of rats and mice indicated an interspecies difference of 1.4 for these two species. This corresponds closely to an interspecies AF solely explained by allometry (7:4 = 1.75) without an additional factor of 2.5 for putative toxicodynamic differences.

Based on the availability of a sufficient toxicity dataset, the default assessment factors (acc. to ECHA GD R8) can be modified into substance specific assessment factors (AF) considering the intrinsic hazard properties of the registered substance. The following findings form the basis of the rationale for the substance specific AF:

In a subchronic feeding study in rats (acc. to OECD TG 408), the primary target organs were the liver and the kidney, i.e. the organs of metabolism and excretion. The hepatic changes were primarily caused by microsomal enzyme induction and are interpreted as an adaptive metabolic response. Changes in the thyroid gland, i.e. presence of altered colloid in line with a reduction of T4 levels can be seen as a consequence of hepatic enzyme induction, as these enzymes are responsible for metabolizing thyroid hormones. The sensitivity of the rat thyroid to increased hepatic clearance of thyroxin is considered to be much higher when compared to humans. The half-life of T4 in rats is lower than in humans (12h vs. 5-9 days respectively), which is likely due to a human specific high-affinity binding globulin for tyroxin, accounting for slower metabolic degradation and clearance. The increased turnover and hepatic clearance of T4 renders the basal activity of the thyroid more active in rats (Meek et al.; Critical Reviews in Toxicology 2003; 33(6), p. 591-653).

The findings in males with respect to kidneys (increased weight and chronic nephropathy) as well as kidney relevant parameters (increased urinary casts) have to be seen in the light of high amounts of alpha2µ-globuline in these animals. Alpha2µ-globuline accumulation is an unique feature of male rats that does not occur in any other species, especially not in male men. Therefore, the findings in kidneys are of doubtful significance to humans.

No effects on reproductive parameters and organs were observed in the repeated dose toxicity study and a multi-generation study with a structurally comparable test substance.

In a gavage developmental toxicity study in rats (acc. to OECD TG 414) adverse effects have been observed consisting of decreased food consumption and decreased body weight gain. Increased liver weights as adaptive response to treatment (enzyme induction) and clinical observations, i.e. temporary salivation, were not assessed as an adverse or toxic effect. The test substance administration had no influence on gestational parameters and induced no adverse signs of developmental toxicity; especially, no indications of teratogenic effects occurred which could be causally related to the test substance administration. In addition, developmental toxicology on a second species, i.e. the rabbit, has been investigated (acc. to OECD TG 414 and GLP). Following dietary treatment, reduced food consumption, reduced body weights, lower body weight gain and/or body weight loss were recorded only for maternal females in the highest dose group tested. Slightly lower fetal body weights in males and females was considered to be secondary to the reduced food intake and markedly decreased body weight gain of the dams. The incidence of unossified metacarpals and/or metatarsals was slightly higher in these fetuses, reflecting the slightly lower fetal weights observed in this dose group. No toxicologically relevant effects on viability, litter size or sex ratio were noted in this study.

In the key studies given above, the nature of effects observed are mainly based on either adaptive, rat specific or unspecific systemic adverse effects such as reduced food consumption and body weight changes. In order to add sufficient conservatism into the DNEL derivation, namely to cover for the uncertainty of an putative systemically toxic parent compound/metabolite being excreted dependent on the caloric demand, an AF of 4 for allometric scaling is included for oral/ dermal systemic long term DNELs.

Since only rat specific but no human relevant organ specific toxicity has been observed, no additional AF covering toxicodynamic differences between rats and humans are considered necessary. In fact, an underestimation of interspecies differences between rats and humans beyond allometric scaling is unlikely due to the favorable toxicological profile of the registered substance.

It needs further to be pointed out, that the multiplicatory principle of different AFs used further adds to conservatism in the derivation of the respective DNELs, especially for the registered substance, which contains a toxicological profile, justifying a non-classification according to 67/548/EEC and regulation (EU) 1272/2008.

 

Substance specific assessment factor for intraspecies extrapolation

Studies on the distribution of human data for various toxicokinetic and toxicodynamic parameters were taken into account, including ‘healthy adults’ of both sexes, young and elderly, mixed races and patients with various medical conditions such as cancer and hypertension. (Hattis 1987, 1999; Hattis and Silver 1994; Renwick and Lazarus, 1998). Using the 95th percentile of the combined distribution of the toxicokinetic and -dynamic variability of datasets is a statistical approach to account for intraspecies variability based on toxicological datasets. On the basis of the above mentioned assessments and statistical approach, an AF of 5 for the general population and AF factor of 3 for the more homogenous worker population can be estimated to account for intraspecies variability.

It needs further to be pointed out, that the multiplicatory principle of AF used further adds to conservatism in the derivation of the respective DNELs, especially for the registered substance, which contains a toxicological profile, justifying a non-classification according to 67/548/EEC and regulation (EU) 1272/2008.

Based on the availability of a sufficient toxicity dataset, the default assessment factors (acc. to ECHA GD R8) can be modified into substance specific assessment factors (AF) considering the intrinsic hazard properties of the registered substance. The following findings form the basis of the rationale for the substance specific AF:

In a subchronic feeding study in rats (acc. to OECD TG 408), the primary target organs were the liver and the kidney, i.e. the organs of metabolism and excretion. The hepatic changes were primarily caused by microsomal enzyme induction and are interpreted as an adaptive metabolic response. Changes in the thyroid gland, i.e. presence of altered colloid in line with a reduction of T4 levels can be seen as a consequence of hepatic enzyme induction, as these enzymes are responsible for metabolizing thyroid hormones. The sensitivity of the rat thyroid to increased hepatic clearance of thyroxin is considered to be much higher when compared to humans. The half-life of T4 in rats is lower than in humans (12h vs. 5-9 days respectively), which is likely due to a human specific high-affinity binding globulin for tyroxin, accounting for slower metabolic degradation and clearance. The increased turnover and hepatic clearance of T4 renders the basal activity of the thyroid more active in rats (Meek et al.; Critical Reviews in Toxicology 2003; 33(6), p. 591-653).

The findings in males with respect to kidneys (increased weight and chronic nephropathy) as well as kidney relevant parameters (increased urinary casts) have to be seen in the light of high amounts of alpha2µ-globuline in these animals. Alpha2µ-globuline accumulation is a unique feature of male rats that does not occur in any other species, especially not in male men. Therefore, the findings in kidneys are of doubtful significance to humans.

No effects on reproductive parameters and organs were observed in the repeated dose toxicity study and a multi-generation study with a structurally comparable test substance.

In a gavage developmental toxicity study in rats (acc. to OECD TG 414) adverse effects have been observed consisting of decreased food consumption and decreased body weight gain. Increased liver weights as adaptive response to treatment (enzyme induction) and clinical observations, i.e. temporary salivation, were not assessed as an adverse or toxic effect. The test substance administration had no influence on gestational parameters and induced no adverse signs of developmental toxicity; especially, no indications of teratogenic effects occurred which could be causally related to the test substance administration. In addition, developmental toxicology on a second species, i.e. the rabbit, has been investigated (acc. to OECD TG 414 and GLP). Following dietary treatment, reduced food consumption, reduced body weights, lower body weight gain and/or body weight loss were recorded only for maternal females in the highest dose group tested. Slightly lower fetal body weights in males and females was considered to be secondary to the reduced food intake and markedly decreased body weight gain of the dams. The incidence of unossified metacarpals and/or metatarsals was slightly higher in these fetuses, reflecting the slightly lower fetal weights observed in this dose group. No toxicologically relevant effects on viability, litter size or sex ratio were noted in this study.

In the key studies given above, the nature of effects observed are mainly based on either adaptive, rat specific or unspecific systemic adverse effects such as reduced food consumption and body weight changes. No human relevant organ specific toxicity is identified for the registered substance, which would justify a conservative default assessment factor for intraspecies variations in toxicokinetics or toxicodynamics. However, an AF of 3 or 5 has been included to cover for remaining uncertainties within a controlled subpopulation, i.e. healthy workers or the general population, respectively.

 

For the worker, the following DNELs were derived:

For derivation of the long-term systemic inhalative DNEL for trans-β-ionone, the oral NOAEL of 72 mg/kg bw/d was taken as a basis and converted into a corrected inhalative NOAEC of 63.5 mg/m3 according to the procedure, recommended in the current guidance document (R8, ECHA 2008). Applying all assessment factors, the inhalative long-term systemic DNEL was set at 12.7mg/m3 for the worker.

 

Long-term – inhalation, systemic effects

Description

Value

Remark

Step 1) Relevant dose-descriptor

NOAEL: 72 mg/kg bw/day

 

Step 2) Modification of starting point

50%/100%

0.38 m3/kg bw

 

 

 

6.7 m3/10 m3

 

Ratio of oral (rat) to inhalation (human) absorption (default value, as proposed in the REACH guidance (R.8.4.2)

Standard respiratory volume of a rat, corrected for 8 h exposure, as proposed in the REACH Guidance (R.8.4.2)

Correction for activity driven differences of respiratory volumes in workers compared to workers in rest (6.7 m3/10 m3)

Modified dose-descriptor

NOAEC corrected inhalative = 72 * (50/100) * (1/0.38) * (6.7/10) = 63.5 mg/m3

Step 3) Assessment factors

 

 

Allometric scaling

1

No allometric scaling has to be applied in case of oral to inhalation route to route extrapolation according to R8 ECHA 2008.

Remaining differences

1

Substance specific assessment factor (see justification above)

Intraspecies

3

Substance specific assessment factor (see justification above)

Exposure duration

2

Use of a subchronic study as starting point forlong-term systemic DNEL derivation (default assessment factor according to R8 ECHA 2008).

Dose response

1

according to R8 ECHA 2008

Quality of database

1

according to R8 ECHA 2008 (GLP guideline Study)

DNEL

Value

 

63.5 / (1 x 1 x 3 x 2 x 1 x 1) = 12.7 mg/m3

 

For derivation of the long-term systemic dermal DNEL for trans-β-ionone, the oral NOAEL of 72 mg/kg bw/d was taken as a basis and was converted into a corrected dermal NOAEL of 144 mg/kg bw/d. Applying all assessment factors, the dermal long-term systemic DNEL derived was 6.0 mg/kg bw/d for the worker.

 

Long-term – dermal, systemic effects 

Description

Value

Remark

Step 1) Relevant dose-descriptor

NOAEL: 72 mg/kg bw/day

 

Step 2) Modification of starting point

2

Substance specific assessment factor for oral to dermal extrapolation based onin vitrodermal penetration/permeability study (see justification above)

Modified dose-descriptor

NOAEL corrected dermal = 72 * 2 = 144 mg/kg bw/d

Step 3) Assessment factors

 

 

Allometric scaling

4

Assessment factor for allometric scaling according to R8 ECHA 2008

Remaining differences

1

Substance specific assessment factor:

(see justification above)

Intraspecies

3

Substance specific assessment factor:

(see justification above)

Exposure duration

2

Use of a subchronic study as starting point forlong-term systemic DNEL derivation (default assessment factor according to R8 ECHA 2008).

Dose response

1

according to R8 ECHA 2008

Quality of database

1

according to R8 ECHA 2008 (GLP guideline Study)

DNEL

Value

 

144 / (4 x 1 x 3 x 2 x 1 x 1) = 6.0 mg/kg bw/day

 

 

No DNELs were derived for local effects after short term or after long term inhalative or dermal exposure and for systemic effects after short term inhalative or dermal exposure, as the substance exhibits no hazardous potential in terms of these endpoints and the conservatively derived long term DNELs for systemic effects covers putative local effects.Trans-β-ionone does not pose any hazard leading to a classification according to the criteria laid down under 67/548/ECC and CLP.

 

General Population - Hazard via inhalation route

Systemic effects

Long term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
3.1 mg/m³
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
Overall assessment factor (AF):
10
Modified dose descriptor starting point:
NOAEC
DNEL value:
31.3 mg/m³
Acute/short term exposure
DNEL related information

Local effects

Acute/short term exposure
DNEL related information

General Population - Hazard via dermal route

Systemic effects

Long term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
3.6 mg/kg bw/day
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
Overall assessment factor (AF):
40
Modified dose descriptor starting point:
NOAEL
DNEL value:
144 mg/kg bw/day
Acute/short term exposure
DNEL related information

General Population - Hazard via oral route

Systemic effects

Long term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
1.8 mg/kg bw/day
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
Overall assessment factor (AF):
40
Modified dose descriptor starting point:
NOAEL
DNEL value:
72 mg/kg bw/day
Acute/short term exposure
DNEL related information

General Population - Hazard for the eyes

Local effects

Hazard assessment conclusion:
no hazard identified

Additional information - General Population

In the 28 day range finding study 5 animals per sex and dose were administered to 1000, 5000 and 15000 ppm (corresponding to ca. 80-100, 360-510 and 1090-1260 mg/kg bw/day). The observed effects were changes in organ weights and food consumption, so that the NOAEL was found to be 80-100 mg/kg bw/day.

Repeated dose toxicity was analyzed in a subchronic 90-days study performed according to OECD Guideline 408 (BASF AG, 2003, 2004). In the subchronic study the compound was administered to groups of 10 male and 10 female Wistar rats at dietary concentrations of 0, 100, 1000 and 10000 ppm (corresponding to 7 and 8 mg/kg bw/day, 72 and 83 mg/kg bw/day or 720 and 801 mg/kg bw/day for males and females, respectively) for 3 months. Substance related effects in liver, kidney and the thyroid gland were detected, including central and peripheral hypertrophy of hepatocytes, decreased thyroxine as well as changes in urinary parameters. Effects observed on reproductive organs were significantly increased absolute and relative weight of the testes and relative weight increase of epididymides in the high dose males, though no histological correlates were detected. The NOEL under the conditions of the present study was 100 ppm for both sexes (about 7 and 8 mg/kg bw/d for males and females). Based on adaptive liver effects in both sexes and minor urine findings in males the NOAEL was 1000 ppm (72 and 83 mg/kg bw/day for males and females, respectively). The LOAEL was found to be 10 000 ppm (720 and 801 mg/kg bw/day for males and females) due to liver, kidney and thyroid findings in both sexes.

In two other studies rats were administered for 5 or 90 days, where a NOAEL of 10 mg/kg bw/day was found after the 90 day application (Hoffmann-LaRoche, 1975; Ford, 1983). However, both studies showed clear deficits in the protocol or given data, so that their results could not be taken into account for evaluation.

 

No adverse effects on reproduction were found in a two-generation study, when 8-10 mg/kg bw/day of the structural analogue mixed ionone isomers (CAS 8013-90-9) were administered to parental rats for 8 months (Sporn, 1963). The F1 generation (offspring) were allowed to reach maturity and were then treated with 15 mg/kg of ionone prior to being subject to reproductive toxicity testing.

 

Developmental toxicity was evaluated in a study performed according to OECD Guideline 414 (BASF AG, 2004). Trans-β-ionone was administered as a solution in olive oil to 25 "time-mated" female Wistar rats/group by stomach tube at doses of 25, 100 and 400 mg/kg bw on day 6 through day 19 post coitum (pc). As a result, administration of 400 mg/kg bw elicited substance-induced effects on the dams including signs of maternal toxicity like reduced body weight gain (-29%). The dosage of 100 mg/kg bw/day resulted in some substance-related findings (i.e. temporary salivation, marginally increased liver weights), which are not considered to be adverse, but mirror some adaptive responses of the animals. The test substance had no influence on gestational parameters and did not induce adverse signs of developmental toxicity or teratogenic effects at all dose levels. The NOAEL for maternal toxicity has been set at 100 mg/kg bw/day and ≥ 400 mg/kg bw/day for developmental toxicity.As the experimental exposure adequately covered the pregnancy of the species under investigation an AF for exposure duration is not necessary.

Information for prenatal developmental toxicity of trans-β-ionone is available on a second species, i.e. the rabbit. In a study performed according to OECD Guideline 414 and GLP (BASF SE, 2014) 22 female New Zealand White rabbits/group received trans-β-ionone by inclusion in the diet on day 6 through day 29 p.c. at target dose levels of 17, 50 and 200 mg/kg bw/day. Following dietary treatment at 200 mg/kg bw/day, reduced food consumption (absolute and relative to body weight) were noted during almost the entire treatment period. In addition, reduced body weights, lower body weight gain and/or body weight loss were recorded. The treatment did not result in mortality or gross findings at necropsy. No maternal toxicity was observed in the 17 and 50 mg/kg bw/day groups.

Fetal body weights were slightly lower in males and females at 200 mg/kg bw/day, reaching statistical significance for males only. This change was considered to be secondary to the reduced food intake and markedly decreased body weight gain of the dams. No toxicologically relevant effects on viability, litter size or sex ratio were noted up to 200 mg/kg bw/day. The incidence of unossified metacarpals and/or metatarsals was slightly higher in the fetuses at 200 mg/kg bw/day, reflecting the slightly lower fetal weights observed in this dose group. The maternal and developmental NOAEL for trans-β-ionone was established as being 50 mg/kg bw/day.

As the experimental exposure of a study according to Guideline OECD 414 adequately covered the pregnancy of the species under investigation an AF for exposure duration is not necessary.

 

Therefore, the respective NOAEL of 72 mg/kg bw/day for female rats in the 90-days toxicity study has been taken as conservative point of departure for the respective systemic DNELs derived, which covers findings observed in the subchronic repeated dose and developmental toxicity study.

 

Route to route extrapolation:

On the basis of the low vapour pressure (0.072 hPa), the exposure with trans-β-ionone via inhalation as a vapour is low. According to Chapter R.8 of REACH Guidance on information requirements and chemical safety assessment, it is proposed in the absence of route-specific information to include a default factor in the case of inhalation-to-oral extrapolation, assuming 50% oral and 100% inhalation absorption.

 

No experimental data on dermal absorption of trans-β-ionone are available. However data from thestructurally similar substance methyl-ionone (CAS 1335-46-2) can be used for read-across.Based on comparable physicochemical properties(M = 192 – 206 g/mol, density = 0.93 – 0.95 g/cm3, logPow= 4 – 5, water solubility < 1 g/l, vapour pressure < 10 Pa) both substances are considered to show a limitedbioavailability via the dermal route. In addition both substances are of low acute toxicity (LD50(dermal and oral) > 5000 mg/kg bw). For the above mentioned reasons, dermal adsorption of both substances is considered to be sufficiently similar to support read-across.

In anin vitrodermal penetration/permeability study, only 0.7% or undetectable amounts of methyl ionone (mixture of isomers) were recovered in the fluid beneath the skin preparations of rats and pigs, respectively, 6 h after application of a 3000 µg dose (600 µg/cm² over 5 cm² of skin) (RIFM, 1984a). In this study, approximately 50% (rat) and 10% (pig) of methyl ionone14C penetrated into, but not through the epidermis and dermis, while another 30% was lost to evaporation. (Belsito, 2007)

Skin penetration potential through human skin is generally much closer to that of porcine skin than to that of rat skin. The morphology of porcine skin corresponds much better to that of human skin than that of fur bearing animals, which presents numerous hair follicles. Hair follicles act as shunt ways of resorption and hence, chemical substances penetrate into or through fur bearing skin much easier. Van Ravenzwaay and Leibold (2004) have compared the differences in absorption for a large number of chemicals and found the dermal absorption through rat skin is generally at least 2.3 times greater than through human skin. Taking this information together with the values of the in vitro study (50% penetration into rat skin and 10% penetration into porcine skin, but no penetration through either kind of skin), a maximal penetration of ca. 20% into but not through the skin might be assumed. A worst case of 50% penetration through human skin is assumed fortrans-β-ionone, although realistic values may be considerably lower. Hence, an assessment factor of 0.5 was chosen.

 

Substance specific assessment factor for remaining differences

Although ‘residual’ interspecies variability may remain following allometric scaling, this is largely accounted for in the assessment factors proposed for intraspecies variability, i.e. reflecting the interdependency of inter- and intraspecies assessment factors (Calabrese and Gilbert, 1993).

Furthermore, within the ERASM project, it was suggested that a factor of 2.5 for ‘remaining‘ interspecies differences may be questionable as a standard procedure (Escher and Mangelsdorf, 2009; Batkeet al, 2010; Bitsch et al, 2006). The comparison of rats and mice indicated an interspecies difference of 1.4 for these two species. This corresponds closely to an interspecies AF solely explained by allometry (7:4 = 1.75) without an additional factor of 2.5 for putative toxicodynamic differences.

Based on the availability of a sufficient toxicity dataset, the default assessment factors (acc. to ECHA GD R8) can be modified into substance specific assessment factors (AF) considering the intrinsic hazard properties of the registered substance. The following findings form the basis of the rationale for the substance specific AF:

In a subchronic feeding study in rats (acc. to OECD TG 408), the primary target organs were the liver and the kidney, i.e. the organs of metabolism and excretion. The hepatic changes were primarily caused by microsomal enzyme induction and are interpreted as an adaptive metabolic response. Changes in the thyroid gland, i.e. presence of altered colloid in line with a reduction of T4 levels can be seen as a consequence of hepatic enzyme induction, as these enzymes are responsible for metabolizing thyroid hormones. The sensitivity of the rat thyroid to increased hepatic clearance of thyroxin is considered to be much higher when compared to humans. The half-life of T4 in rats is lower than in humans (12h vs. 5-9 days respectively), which is likely due to a human specific high-affinity binding globulin for tyroxin, accounting for slower metabolic degradation and clearance. The increased turnover and hepatic clearance of T4 renders the basal activity of the thyroid more active in rats (Meek et al.; Critical Reviews in Toxicology 2003; 33(6), p. 591-653).

The findings in males with respect to kidneys (increased weight and chronic nephropathy) as well as kidney relevant parameters (increased urinary casts) have to be seen in the light of high amounts of alpha2µ-globuline in these animals. Alpha2µ-globuline accumulation is an unique feature of male rats that does not occur in any other species, especially not in male men. Therefore, the findings in kidneys are of doubtful significance to humans.

No effects on reproductive parameters and organs were observed in the repeated dose toxicity study and a multi-generation study with a structurally comparable test substance.

In a gavage developmental toxicity study in rats (acc. to OECD TG 414) adverse effects have been observed consisting of decreased food consumption and decreased body weight gain. Increased liver weights as adaptive response to treatment (enzyme induction) and clinical observations, i.e. temporary salivation, were not assessed as an adverse or toxic effect. The test substance administration had no influence on gestational parameters and induced no adverse signs of developmental toxicity; especially, no indications of teratogenic effects occurred which could be causally related to the test substance administration. In addition, developmental toxicology on a second species, i.e. the rabbit, has been investigated (acc. to OECD TG 414 and GLP). Following dietary treatment, reduced food consumption, reduced body weights, lower body weight gain and/or body weight loss were recorded only for maternal females in the highest dose group tested. Slightly lower fetal body weights in males and females was considered to be secondary to the reduced food intake and markedly decreased body weight gain of the dams. The incidence of unossified metacarpals and/or metatarsals was slightly higher in these fetuses, reflecting the slightly lower fetal weights observed in this dose group. No toxicologically relevant effects on viability, litter size or sex ratio were noted in this study.

In the key studies given above, the nature of effects observed are mainly based on either adaptive, rat specific or unspecific systemic adverse effects such as reduced food consumption and body weight changes. In order to add sufficient conservatism into the DNEL derivation, namely to cover for the uncertainty of an putative systemically toxic parent compound/metabolite being excreted dependent on the caloric demand, an AF of 4 for allometric scaling is included for oral/ dermal systemic long term DNELs.

Since only rat specific but no human relevant organ specific toxicity has been observed, no additional AF covering toxicodynamic differences between rats and humans are considered necessary. In fact, an underestimation of interspecies differences between rats and humans beyond allometric scaling is unlikely due to the favorable toxicological profile of the registered substance.

It needs further to be pointed out, that the multiplicatory principle of different AFs used further adds to conservatism in the derivation of the respective DNELs, especially for the registered substance, which contains a toxicological profile, justifying a non-classification according to 67/548/EEC and regulation (EU) 1272/2008.

 

Substance specific assessment factor for intraspecies extrapolation

Studies on the distribution of human data for various toxicokinetic and toxicodynamic parameters were taken into account, including ‘healthy adults’ of both sexes, young and elderly, mixed races and patients with various medical conditions such as cancer and hypertension. (Hattis 1987, 1999; Hattis and Silver 1994; Renwick and Lazarus, 1998). Using the 95th percentile of the combined distribution of the toxicokinetic and -dynamic variability of datasets is a statistical approach to account for intraspecies variability based on toxicological datasets. On the basis of the above mentioned assessments and statistical approach, an AF of 5 for the general population and AF factor of 3 for the more homogenous worker population can be estimated to account for intraspecies variability.

It needs further to be pointed out, that the multiplicatory principle of AF used further adds to conservatism in the derivation of the respective DNELs, especially for the registered substance, which contains a toxicological profile, justifying a non-classification according to 67/548/EEC and regulation (EU) 1272/2008.

Based on the availability of a sufficient toxicity dataset, the default assessment factors (acc. to ECHA GD R8) can be modified into substance specific assessment factors (AF) considering the intrinsic hazard properties of the registered substance. The following findings form the basis of the rationale for the substance specific AF:

In a subchronic feeding study in rats (acc. to OECD TG 408), the primary target organs were the liver and the kidney, i.e. the organs of metabolism and excretion. The hepatic changes were primarily caused by microsomal enzyme induction and are interpreted as an adaptive metabolic response. Changes in the thyroid gland, i.e. presence of altered colloid in line with a reduction of T4 levels can be seen as a consequence of hepatic enzyme induction, as these enzymes are responsible for metabolizing thyroid hormones. The sensitivity of the rat thyroid to increased hepatic clearance of thyroxin is considered to be much higher when compared to humans. The half-life of T4 in rats is lower than in humans (12h vs. 5-9 days respectively), which is likely due to a human specific high-affinity binding globulin for tyroxin, accounting for slower metabolic degradation and clearance. The increased turnover and hepatic clearance of T4 renders the basal activity of the thyroid more active in rats (Meek et al.; Critical Reviews in Toxicology 2003; 33(6), p. 591-653).

The findings in males with respect to kidneys (increased weight and chronic nephropathy) as well as kidney relevant parameters (increased urinary casts) have to be seen in the light of high amounts of alpha2µ-globuline in these animals. Alpha2µ-globuline accumulation is a unique feature of male rats that does not occur in any other species, especially not in male men. Therefore, the findings in kidneys are of doubtful significance to humans.

No effects on reproductive parameters and organs were observed in the repeated dose toxicity study and a multi-generation study with a structurally comparable test substance.

In a gavage developmental toxicity study in rats (acc. to OECD TG 414) adverse effects have been observed consisting of decreased food consumption and decreased body weight gain. Increased liver weights as adaptive response to treatment (enzyme induction) and clinical observations, i.e. temporary salivation, were not assessed as an adverse or toxic effect. The test substance administration had no influence on gestational parameters and induced no adverse signs of developmental toxicity; especially, no indications of teratogenic effects occurred which could be causally related to the test substance administration. In addition, developmental toxicology on a second species, i.e. the rabbit, has been investigated (acc. to OECD TG 414 and GLP). Following dietary treatment, reduced food consumption, reduced body weights, lower body weight gain and/or body weight loss were recorded only for maternal females in the highest dose group tested. Slightly lower fetal body weights in males and females was considered to be secondary to the reduced food intake and markedly decreased body weight gain of the dams. The incidence of unossified metacarpals and/or metatarsals was slightly higher in these fetuses, reflecting the slightly lower fetal weights observed in this dose group. No toxicologically relevant effects on viability, litter size or sex ratio were noted in this study.

In the key studies given above, the nature of effects observed are mainly based on either adaptive, rat specific or unspecific systemic adverse effects such as reduced food consumption and body weight changes. No human relevant organ specific toxicity is identified for the registered substance, which would justify a conservative default assessment factor for intraspecies variations in toxicokinetics or toxicodynamics. However, an AF of 3 or 5 has been included to cover for remaining uncertainties within a controlled subpopulation, i.e. healthy workers or the general population, respectively.

 

For the general population, the following DNELs were derived:

For derivation of the long-term systemic inhalative DNEL for trans-β-ionone, the oral systemic no adverse effect level, i.e. 72 mg/kg bw/d was taken as a basis and converted into a corrected inhalative NOAEC of 31.3 mg/m3 according to the procedure, recommended in the current guidance document (R8, ECHA 2008). Applying all assessment factors, the inhalative long-term systemic DNEL was set at 3.1mg/m3 for the general population.

 

Long-term – inhalation, systemic effects

Description

Value

Remark

Step 1) Relevant dose-descriptor

NOAEL: 72 mg/kg bw/day

 

Step 2) Modification of starting point

50%/100%

 

 

 

1.15 m3/kg bw 

Ratio of oral (rat) to inhalation (human) absorption (default value, as proposed in the REACH guidance (R.8.4.2)

 

Standard respiratory volume of a rat, corrected for 24 h exposure, as proposed in the REACH Guidance (R.8.4.2)

Modified dose-descriptor

NOAEC corrected inhalative = 72 * (50/100) *(1/1.15)= 31.3 mg/m3

Step 3) Assessment factors

 

 

Allometric scaling

1

No allometric scaling has to be applied in case of oral to inhalation route to route extrapolation according to R8 ECHA 2008.

Remaining differences

1

Substance specific assessment factor (see justification above)

Intraspecies

5

Substance specific assessment factor (see justification above)

Exposure duration

2

Use of a subchronic study as starting point forlong-term systemic DNEL derivation (default assessment factor according to R8 ECHA 2008).

Dose response

1

according to R8 ECHA 2008

Quality of database

1

according to R8 ECHA 2008 (GLP guideline Study)

DNEL

Value

 

31.3 / (1 x 1 x 5 x 2 x 1 x 1) = 3.1 mg/m3

 

For derivation of the long-term systemic dermal DNEL for trans-β-ionone,the oral NOAEL of 72 mg/kg bw/d was taken as a basis and was converted into a corrected dermal NOAEL of 144 mg/kg bw/d. Applying all assessment factors, the dermal long-term systemic DNEL derived was 3.6 mg/kg bw/d for thegeneral population.

 

Long-term – dermal, systemic effects 

Description

Value

Remark

Step 1) Relevant dose-descriptor

NOAEL: 72 mg/kg bw/day

 

Step 2) Modification of starting point

2

Substance specific assessment factor for oral to dermal extrapolation based onin vitrodermal penetration/permeability study (see justification above)

Modified dose-descriptor

NOAEL corrected dermal = 72 * 2 = 144 mg/kg bw/d

Step 3) Assessment factors

 

 

Allometric scaling

4

Assessment factor for allometric scaling according to R8 ECHA 2008

Remaining differences

1

Substance specific assessment factor:

(see justification above)

Intraspecies

5

Substance specific assessment factor:

(see justification above)

Exposure duration

2

Use of a subchronic study as starting point forlong-term systemic DNEL derivation (default assessment factor according to R8 ECHA 2008).

Dose response

1

according to R8 ECHA 2008

Quality of database

1

according to R8 ECHA 2008 (GLP guideline Study)

DNEL

Value

 

144 / (4 x 1 x 5 x 2 x 1 x 1) = 3.6 mg/kg bw/day

 

For derivation of the long-term systemic oral DNEL for trans-β-ionone, the oral NOAEL of 72 mg/kg bw/d was taken as a basis. After applying the assessment factors, the oral long-term systemic DNEL was set at 1.8 mg/kg bw/d for the general population.

 

Long-term – oral, systemic effects

Description

Value

Remark

Step 1) Relevant dose-descriptor

NOAEL: 72 mg/kg bw/day

 

Step 2) Modification of starting point

-

-

Step 3) Assessment factors

 

 

Allometric scaling

4

Assessment factor for allometric scaling according to R8 ECHA 2008

Remaining differences

1

Substance specific assessment factor:

(see justification above)

Intraspecies

5

Substance specific assessment factor:

(see justification above)

Exposure duration

2

Use of a subchronic study as starting point forlong-term systemic DNEL derivation (default assessment factor according to R8 ECHA 2008).

Dose response

1

according to R8 ECHA 2008

Quality of database

1

according to R8 ECHA 2008 (GLP guideline Study)

DNEL

Value

 

72 / (4 x 1 x 5 x 2 x 1 x 1) = 1.8 mg/kg bw/day

 

 

Consumer is exposed toβ-ionone in food, cosmetics and some house wares like cleaning agents (flavoring compound, aroma additive). In general, consumer exposure is low since only small amounts ofβ-ionone are contained in cosmetics at usual concentrations of up to 0.3 % and in food in maximum amounts ranging from 0.5 – 10 ppm. β-Ionone was granted GRAS status by FEMA (in 1965) and is approved by the FDA for food use (21 CFR 121.1164) (cited in Opdyke, 1979). The council of Europe (EU, 1999) listedβ-ionone giving ADI of 0.1 mg/kg bw/day. The Joint FAO/WHO Expert Committee on Food Additives (JECFA, 2004) has published a monograph and specification, giving conditional ADI of 0 - 0.1 mg/kg bw/day. The Canadian Food Inspection Agency (Feed Section) listedβ-ionone as approved feed ingredient with a maximum limit of 12.5 ppm (CFIA, 2004). The Japan Flavor & Fragrance Material Association putβ-ionone on their list of designated additives-flavorings as a flavoring used for food in Japan without any details of permitted uptake levels (FFCR Japan, 2004). According to Opdyke (1979) typical use concentrations in final products are 0.03% (soap), 0.003% (detergent), 0.016% (creams, lotions) and 0.3% (perfume). The odor threshold is indicated with 0.007 ppb or 56 ng/m³ based on vapor (Roempp, 2004) and 1μg/l based on the concentration in water (subjectively determined (Wilkesmann et al., 1995)).

The maximum skin level that results from the use of trans-β-ionone in formulae that go into fine fragrances has been reported to be 1.46% (IFRA, 2001), assuming use of the fragrance oil at levels up to 20% in the final product. The 97.5th percentile use level in formulae for use in cosmetics in general has been reported to be 3.11% (IFRA, 2001), which would result in a conservative calculated maximum daily exposure on the skin of 0.08 mg/kg for high end users of these products.

 

No DNELs were derived for local effects after short term or after long term inhalative or dermal exposure and for systemic effects after short term inhalative, dermal or oral exposure, as the substance exhibits no hazardous potential in terms of these endpoints and the conservatively derived long term DNELs for systemic effects covers putative local effects. Trans-β-iononedoes not pose any hazard leading to a classification according to the criteria laid down under 67/548/ECC and CLP.