Fatty acids, C16-18 and C18-unsatd., reaction products with diethylenetriamine, di-Me sulfate-quaternized

Toxicological Summary

Workers - Hazard via inhalation route

Systemic effects

Long term exposure

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

44 mg/m³

Most sensitive endpoint:

repeated dose toxicity

DNEL related information

Overall assessment factor (AF):

12

Modified dose descriptor starting point:

NOAEC

Acute/short term exposure

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

132 mg/m³

Most sensitive endpoint:

repeated dose toxicity

DNEL related information

Local effects

Long term exposure

Hazard assessment conclusion:

no-threshold effect and/or no dose-response information available

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:

12.5 mg/kg bw/day

Most sensitive endpoint:

repeated dose toxicity

DNEL related information

Overall assessment factor (AF):

24

Modified dose descriptor starting point:

NOAEL

Acute/short term exposure

DNEL related information

Local effects

Long term exposure

Hazard assessment conclusion:

no-threshold effect and/or no dose-response information available

Additional information - workers

Table: Critical DN(M)ELs for workers

Exposure pattern	Route	Descriptors^1	DNEL/DMEL (appropriate unit)	Most sensitive endpoint
Acute - systemic effects	dermal (mg/kg bw /day)	DNEL	NA	NA
	Inhalation (mg/m^3)	DNEL	132 mg/m^3/15 min*	Acute toxicity (calculated)
Acute - local effects	Dermal (mg/cm^2)	DNEL	NA	NA
	Inhalation (mg/m^3)	Not quantifiable	NA	NA
Long-term - systemic effects	Dermal (mg/kg bw /day)	DNEL	12.5 mg/kg bw/d	Repeated dose toxicity
	Inhalation (mg/m^3)	DNEL	44 mg/m^3(8h – TWA)	Repeated dose toxicity (calculated)
Long-term – local effects	Dermal (mg/cm^2)	Not quantifiable	NA	NA
	Inhalation (mg/m^3)	Not quantifiable	NA	NA

* calculated as 3 times the value (default 3) of the long-term DNEL (44 mg/m³ x 3)

[1] Values in IUCLID 5 are DNEL/DMEL/ not quantifiable

Worker-DNEL acute systemic effects:

A DNEL for acute toxicity is not established for the dermal and the oral route, because no acute toxicity hazard leading to classification and labelling has been identified, and there is no potential for high peak exposures.

The most likely route of human exposure for workers and consumers is the dermal route. Systemic levels of the substance will be low due to the very low dermal absorption rate of a maximum of 1% for this substance class (section 5.1, Toxicokinetics). Furthermore, its irritating properties will require the use of protective industrial hygiene measures to keep the skin contact with the substance to a minimum. Therefore, the dermal route of application is assessed as not relevant for systemic exposure. A dermal DNEL longterm systemic has been derived by route to route extrapolation from oral uptake using conservative assessment factors for oral and dermal uptake.

DNEL acute inhalation worker

High peak exposures are usually relevant for the inhalation route only. Vaporisation needs not to be considered due to the very low vapour pressure of approx.7.5 x 10E-22 Pa. The generation and effects of inhalable particles or droplets such as dust or aerosols is considered negligible under the specific operational conditions for this substance.

For an assessment of potential respiratory irritation upon inhalation of aerosols, test results with a diluted test substance (5% of active ingredient) are taken as a point of reference. In these studies only mild irritative effects to the eye have been shown which did not result in classification as an eye irritant and can be regarded as a threshold value for mucous membrane irritation.

The DNEL for acute inhalation toxicity was set for a reference period of 15 minutes at 3 times the value (default 3) of the long-term DNEL. This approach is appropriate because similar mechanisms of actions are probably involved in the responses to single and repeated exposure (TGD R8).

Local irritation of the respiratory tract

Inhalation studies are not available. A reliable dose descriptor for local irritation of the respiratory tract could not be derived from the available studies. From eye irritation studies it was concluded that irritation to mucuous membranes is at most only slight with a 75% substance preparation not leading to a classification of the test substance for eye irritation. Below a threshold value of 5% test substance no eye irritation was observed. Based on the very low vapour pressure of the test substance and the absence of aerosols either as dusts or mists under normal handling conditions, irritation to the upper respiratory tract can be practically excluded.

Local effects dermal

Local irritation of the skin can occur after acute and long-term exposure to undiluted IQACs. Tests with diluted substances show that a specific concentration limit can be established for preparations with 5% active ingredient. Only mild irritative effects to the skin have been reported for this substance concentration.

Worker-DNEL long-term for dermal route (systemic)

The RA concluded that long-term oral repeated exposure to IQACs might have the potential to cause mild systemic effects on the liver accompanied with decreased serum albumin concentration and increased levels of serum glutamic pyruvic and serum glutamic oxaloacetic transaminase. The relevance of these findings is low due to the absence of histopathological correlates. These effects were only seen in male animals at the highest dose tested. Moreover, the dose setting with intervals of a factor of ten in this study does not comply with the current dose setting strategy. Therefore, a NOAEL has been calculated from the highest dose level as the LOAEL with an Assessment Factor of 3 instead of departing from the reported NOEL of 100 mg/kg bw/d, which was the middle dose level in this study. On this basis a dose descriptor has been determined from the 91-day oral study on rats taking a NOAEL of 300 mg/kg bw/d.

Though a subchronic dermal toxicity study conducted in rabbits for 91 days has been identified (NICNAS 1999), this study was regarded as inadequate for an assessment of systemic toxicity induced by dermal exposure. The reason being, that it has been conducted with only two dose groups and was limited to low doses due to irritative skin effects. In order to characterise the dose-response relationship for systemic effects via the dermal route it was therefore necessary to obtain a long-term dermal DNEL by route-to-route extrapolation.

According to NICNAS (1999) toxicokinetic data show low oral uptake judged by intestinal absorption performed on bile duct canulated rats based on a study that was conducted with the objective to distinguish if the substance was not absorbed at all or if it was subject to biliary excretion. According to the report, excretion with the bile proved not to be a relevant route as over 72 hours only 0.07% of the material was detected in the bile. Consequently, it can be confirmed that the oral bioavailability of IQACs is minimal, and can be expected to be far less than 1% (Ref.: Procter & Gamble, 1979; TheProcter & Gamble Company (1979): Metabolism Screen – MTBL (rats; modified P&G standard procedure 23; oral dosing with bile duct canulation; being prepared by MVL).

In a study with radiolabelled Oleic-acid based IQAC, DMS quaternised CAS-No. 72749-55-4) the intestinal absorption was studied for 96 hrs after application to female rats (Wistar SPF-Cpb) after a single oral dose of approx.10 mg/kg bw.The 14C radiolabel was in the N-methyl group of the imidazolinium ring. After 96 hrs an intestinal absorption of approx. 0.79% of the administered dose was found (Henkel 1986a).

In a study with radiolabelled Oleic-acid based IQAC, DMS quaternised CAS-No. 72749-55-4) the dermal absorption was studied by dermal application to female rats (Wistar SPF-Cpb) after a single dermal dose in two test groups of 5 and 8 females (body weights approx. 222 and 241 gm, respectively, in groups 1 and 2). The 14C-radiolabel was in the N-methyl group of the imidazolinium ring. Approximately 200 mg of the compound solution was applied cutaneously as solution in water at a concentration of 0.1% (group 1) and 0.5% (group 2) for 48 hrs under non-occlusive conditions. The application area of 10 cm2 was covered with a glass capsule, cemented in place, which allowed gas exchange with the ambience but impeded oral uptake. In the 48 hrs exposure period less than 0.51 % (group1) and 2-3% (group2) were absorbed through the skin of the rats. The latter uptake rate occurred in the presence of irritative skin damage with accompanying significant scatter of data (Henkel 1986b).

As reported by NICNAS (1999) three male Sprague Dawley rats (body weight 196-201 gm) were treated topically with radiolabelled [N-14C-methyl-labelled] fully saturated IQAC, DMS quaternised (CAS-No. 72623-82-6), radiochemical purity: 98.8 %.

Each animal received a dose of 5.9 mg/kg bw in approximately 0.1 ml of water, corresponding to approx.14 µCi per animal. Animals were housed in metabolic cages, and sacrificed 72 hours after treatment. 14C was analysed upon necropsy in a wide range of organs including skin. Analyses were also performed on urine, faeces, expired CO2, blood, and plasma. Furthermore, cage washes and residual activity in dosing syringes were monitored.

Following topical administration to the skin of rats of radiolabelled test material, 89% of the cutaneously applied dose remained at the test site. The carcass contained a mere 0.02% of the 14C radioactivity, and excretion accounted for 0.03% of the dose with the urine, and 0.03% of the dose with the faeces. Detectable, but low radioactivity was present in only two other tissues, adjacent skin (0.0002% of dose) and bone marrow (0.02 µg/g; Ref.: The Procter & Gamble Company (1978): Metabolism Screen – MTBL (rats; P&G standard procedure 23; dermal dosing).

Regarding the toxicokinetic behaviour of IQAC compounds, the above cited studies show that there are very low levels of absorption of the test substance from the gastrointestinal tract or dermally. The small amounts absorbed were rapidly excreted. One can conclude from these studies that imidazolium quaternary ammonium compounds do not exhibit any significant systemic accumulation following repeated ingestion or dermal contact (The Procter & Gamble Company (1978): Metabolism Screen – MTBL (rats; P&G standard procedure 23; dermal dosing).

As a Point of Departure for oral and dermal uptake an upper worst case rate of 1% is taken forward to the DNEL derivations.

The dermal NOAEL is therefore determined to be equal to the oral calculated NOAEL, i.e. approx. 300 mg/kg bw/day.

Table: Assessment factors and DNEL calculation for worker DNEL long-term dermal systemic effects

Uncertainties	AF	Justification
interspecies differences;	4	The starting point is an oral dose descriptor from a rat study. It is therefore necessary to include an allometric scaling factor of 4 to take account of differences in basal metabolic rates between rats and humans. There are no data for IQACs to quantify other differences between animals and humans that could affect extrapolation. On the basis of the very low systemic absorption rate of the test substance, the default assumption of a worst case maximum absorption of 1% and the availability of toxicokinetic and toxicodynamic data no further AF is deemed necessary to be included in the interspecies extrapolation giving an overall assessment factor of 4.
intraspecies differences;	3	There are no data to quantify variability in susceptibility to the effects of long-term exposure to IQACs in the human population. The default factor of 3 for workers will therefore be used to take account of intraspecies variability.
differences in duration of exposure;	2	Sub-chronic to chronic
Dose response and endpoint specific/severity	1	Effects on the male rat liver at the highest dose tested without histopathological correlate and potentially reversible changes in serum levels of albumin, SGPT and SGOT, being the only systemic substance induced effects identified.
quality of whole database.	1	The key studies were conducted to modern regulatory standards apart from the dose spacing by a factor of ten. Therefore a calculated NOAEL has been derived which would be more appropriate to modern regulatory standards. The DNEL derived from the subchronic oral study in rats therefore departs from the LOAEL of approx. 900 mg/kg bw/d (rel. to active ingredient) and leads to a calculated NOAEL of 300 mg/kg bw/ d (rel. to a.i.)
Overall AF: 24 (300)
Endpoint specific DNEL (from 91-day study)			300/24 = 12.5 mg/kg bw/d

Worker-DNEL long-term for inhalation route (systemic)

No studies have been undertaken by inhalation to characterise the dose-response relationship for systemic effects. Therefore, it will be necessary to obtain a long-term inhalation DNEL by route-to-route extrapolation. Human data is not available.

The NOAEL for systemic toxicity identified from an oral 91-day-study in the rat was 300 mg/kg bw/d. As has been already discussed, a 1 % oral absorption, based on toxicokinetic data is taken forward for IQACs for a route-to-route extrapolation. As a worst-case assumption a 1% absorption after inhalation exposure is assumed, as the available toxicodynamic data have shown a generally low membrane penetration rate (intestinal / dermal) independent of the route of exposure. Therefore, for inhalation the assumption of a similar low absorption rate appears appropriate. Nevertheless, a default AF of 2 for route extrapolation (oral -> inhalation) to adjust the NOAEC for bioavailability is included, though toxicokinetic and toxicodynamic date indicate a similar low absorption through biological membrane barriers.

For the derivation of a NOAEC for the worker the following corrections have to be applied to the oral NOAEL (rat). The oral NOAEL (rat) is multiplied with 1/0.38 m³/kg bw/8h (default respiratory volume in rat, Table R.8.2 of CSR guidance) to give the corresponding rat inhalation 8h-NOAEC (no-observed adverse effect concentration). To obtain the starting point for workers, a factor of 0.67 is applied to the NOAEC to account for the differences in inhalation rates between animals at rest and humans involved in light activity.

For workers the corrected inhalation NOAEC is calculated according to the following equation:

corrected inhalation NOAEC = oral NOAEL x 1/sRV_ratx ABS_oral-rat/ ABS_inh-humanx sRV_human/ wRV¹

= 300 x 1/0.38 x 6.7/10

The corrected inhalation NOAEC_worker(8h) is therefore:

= 528 mg/m³(8h-TWA)

Adjustment for route extrapolation (oral -> inhalation) is made by introducing an AF of 2 (see below)

Table: Assessment factors and DNEL calculation for worker DNEL long-term inhalation systemic effects

Uncertainties	AF	Justification
interspecies differences;	1	It is not necessary to apply an allometric scaling factor because the starting point has been corrected for differences in respiratory volume and this takes account of differences in metabolic rates. On the basis of the very low systemic absorption rate of the test substance, the default assumption of a worst case maximum absorption of 1% and the availability of toxicokinetic and toxicodynamic data no further AF is deemed necessary to be included in the interspecies extrapolation.
intraspecies differences	3	There are no data to quantify variability in susceptibility to the effects of long-term exposure to IQACs in the human population. The default factor of 3 for workers will therefore be used to take account of intraspecies variability.
differences in duration of exposure	2	It is expected that the severity of effects could increase with duration. Since the dose descriptor is derived from a 91-day study, it is necessary to apply a factor of 2 to take account of extrapolation of subchronic data from the 91-day study to chronic exposure.
Dose response and endpoint specific/severity	1	The effects seen in the 91-day study are only minor toxicological effects. The possibly substance related effects in high dose males with respect to changes in liver weight without histopathological correlate and increased serum albumin and transaminase activity are regarded as slight and probably reversible. It is therefore not necessary to apply a factor to take account of this.
quality of whole database.	1	The key studies were conducted according to modern regulatory standards and were adequately reported. Furthermore, detailed data on toxicokinetics and toxicodynamic properties are available.
Adjustment for bioavailability	2	A default AF of 2 for route extrapolation (oral -> inhalation) to adjust the NOAEC for bioavailability is included, though toxicokinetic and toxicodynamic date indicate a similar low absorption through biological membrane barriers.
Overall AF: 12
Endpoint specific DNEL (from 91-day study)			528/12 = 44 mg/m^3(8h-TWA)

[1] ABS: Absorption; sRV: standard Respiratory Volume; wRV: worker Respiratory Volume

General Population - Hazard via inhalation route

Systemic effects

Long term exposure

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

13 mg/m³

Most sensitive endpoint:

repeated dose toxicity

DNEL related information

Overall assessment factor (AF):

20

Modified dose descriptor starting point:

NOAEC

Acute/short term exposure

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

13 mg/m³

Most sensitive endpoint:

repeated dose toxicity

DNEL related information

Overall assessment factor (AF):

3

Modified dose descriptor starting point:

NOAEC

Local effects

Long term exposure

Hazard assessment conclusion:

no-threshold effect and/or no dose-response information available

Acute/short term exposure

Hazard assessment conclusion:

no-threshold effect and/or no dose-response information available

DNEL related information

General Population - Hazard via dermal route

Systemic effects

Long term exposure

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

7.5 mg/kg bw/day

Most sensitive endpoint:

repeated dose toxicity

DNEL related information

Overall assessment factor (AF):

40

Modified dose descriptor starting point:

NOAEL

Acute/short term exposure

Hazard assessment conclusion:

no-threshold effect and/or no dose-response information available

DNEL related information

Local effects

Long term exposure

Hazard assessment conclusion:

no-threshold effect and/or no dose-response information available

Acute/short term exposure

Hazard assessment conclusion:

no-threshold effect and/or no dose-response information available

General Population - Hazard via oral route

Systemic effects

Long term exposure

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

7.5 mg/kg bw/day

Most sensitive endpoint:

repeated dose toxicity

DNEL related information

Overall assessment factor (AF):

40

Modified dose descriptor starting point:

NOAEL

Acute/short term exposure

Hazard assessment conclusion:

no-threshold effect and/or no dose-response information available

DNEL related information

Additional information - General Population

DNEL acute systemic effects:

A DNEL for acute toxicity is not established for the dermal and the oral route, because no acute toxicity hazard leading to classification and labelling has been identified, and there is no potential for high peak exposures.

Inhalation exposure via liquid aerosols can be excluded, as applications in the form of "rinse-of" aids are under low pressure and unlikely to generate inhalable mists.

The DNEL for acute inhalation toxicity was set for a reference period of 15 minutes at 3 times the value (default 3) of the long-term DNEL. This approach is appropriate because similar mechanisms of actions are probably involved in the responses to single and repeated exposure.

General population-DNEL long-term for dermal route (systemic)

The long term DNEL for general population was derived from the oral 91-day oral study with rats (NOAEL 300 mg/kg bw/d) by route to route extrapolation with a default of 1. The following assessment factors were applied: 4 for interspecies variability, 5 for intraspecies variability general population and 2 for time extrapolation. This results in an overall assessment factor of 40.

Endpoint specific DNEL (from 91-day study)

300/40 = 7.5 mg/kg bw/d

General population-DNEL long-term for inhalation route (systemic)

Concerning route-to-route extrapolation see discussion above (worker).

For the derivation of a NOAEC for the general population the following corrections have to be applied to the oral NOAEL (rat). The oral NOAEL (rat) of 300 mg/kg bw/d is multiplied with 1/1.15 m³/kg bw/d (default respiratory volume in rat, Table R.8.2 of CSR guidance) to give the corresponding rat inhalation NOAEC (no-observed adverse effect concentration). No adjustment of the NOAEC for bioavailability is performed because toxicokinetic and toxicodynamic data indicate a similar low absorption through biological membrane barriers.

For general population in case of 24h exposure/d the corrected inhalation NOAEC is calculated according to the following equation:

corrected inhalation NOAEC = oral NOAEL x 1/sRV_rat¹

= 300 x 1/1.15

The corrected inhalation NOAEC_{general population}(24h) is therefore:

= 260 mg/m³

The following assessment factors were applied: 1 for interspecies variability, 5 for intraspecies variability general population, default AF of 2 for route extrapolation (oral -> inhalation) to adjust the NOAEC for bioavailability and 2 for time extrapolation. This results in an overall assessment factor of 20.

Endpoint specific DNEL (from 91-day study)

260/20 = 13 mg/m^3

General population-DNEL long-term for oral route (systemic)

The long term DNEL for general population was derived from the oral 91-day oral study with rats (NOAEL 300 mg/kg bw/d). The following assessment factors were applied: 4 for interspecies variability, 5 for intraspecies variability general population and 2 for time extrapolation. This results in an overall assessment factor of 40.

Endpoint specific DNEL (from 91-day study)

300/40 = 7.5 mg/kg bw/d

Overall Discussion – Worker and general population:

Reproductive Toxicity: Fertility

Concerning fertility there are no animal studies specifically investigating this endpoint. However, no effects on organ weights of ovary and testes and histopathology of gonads from the 91-day repeated dose study were detected with doses of up to 1000 mg/kg bw/d. There is no information available in humans.

The key study used for the derivation of a DNEL fertility is the 91 day repeated dose toxicity study with a NOAEL of 1000 mg/kg bw/d for effects on fertility.

The following assessment factors were applied: 4 for interspecies variability, 5 (3) for intraspecies variability general population (worker) and 2 for time extrapolation subchronic to chronic. Due to the higher degree uncertainty of available data, which is driven by the fact that only a repeated dose toxicity study was available for assessing effects on fertility, an additional assessment factor of 2 was applied. This results in an overall assessment factor of 80 (48).

For inhalation exposure, the AF for interspecies variability is 1, as the Point of Departure is human inhalation exposure assessment. An additional default AF of 2 is introduced, instead, to account for bioavailability by oral to inhalation route extrapolation, though toxicokinetic and toxicodynamic data suggest a similar low absorption via biological membranes. An AF of 5 accounts for intraspecies variability general population (AF of 3 for workers) and an AF of 2 due to the higher degree of uncertainty of available data which is driven by the fact that only a repeated dose toxicity study was available for assessing effects on fertility. This results in an overall assessment factor of 24 (40) for inhalation exposure for workers (general population) for the endpoint fertility.

Table: DNELs Fertility Workers and General Population

DNEL worker fertility dermal	1000/48 = 20.8 mg/kg bw/day
DNEL worker fertility inhalation	1763/24 = 73.4 mg/m^3/8h-TWA
DNEL general population fertility dermal	1000/80 = 12.5 mg/kg bw/day
DNEL general population fertility oral	1000/80 = 12.5 mg/kg bw/day
DNEL general population fertility inhalation	869/40 = 21.7 mg/m^3/day

The DNELs derived from this study by applying an additional safety factor of 2 to account for higher uncertainty are higher than the DNELs for repeated dose toxicity. Therefore the DNELs for repeated dose toxicity (oral, dermal and inhalation) are also protective for fertility.

Reproductive Toxicity: Developmental toxicity

Concerning developmental toxicity there is one study available covering this endpoint. In this study no effects on maternal reproduction, embryo lethality, or developmental effects were observed following maternal exposure up to 1000 mg/kg bw/day.

A DNEL derived from these studies will be higher than the DNEL derived for repeated dose toxicity as no special assessment factor for exposure time ((‘informed’ AF of 1)) will be introduced (critical time window of effects has been covered in this study). Therefore the DNELs for repeated dose toxicity (oral, dermal and inhalation) are also protective for developmental toxicity.

Carcinogenicity

The mutagenic and clastogenic potential of IQACs has been evaluated in the full range of in vitro and in a single in vivo genotoxicity studies. There was no evidence for genotoxic properties of any of the investigated substances. Although carcinogenicity studies are not available for this structure family, the absence of genotoxicity or inflammatory responses in repeated dose toxicity studies do no raise any specific concerns with regard to carcinogenicity.

[1] ABS: Absorption; sRV: standard Respiratory Volume; wRV: worker Respiratory Volume